CA2216868A1 - High efficiency ex vivo transduction of hematopoietic stem cells by recombinant retroviral preparations - Google Patents

Abstract

Compositions and methods for the efficient ex vivo introduction of nucleic acid into hematopoietic stem cells mediated by recombinant retrovirus particles is described. The recombinant vector constructs carried by the recombinant retrovirus particles code for the production of a desired gene product having a therapeutic application from a gene of interest. Upon re-introduction into a patient, the transduced hematopoietic stem cells produce a desired gene product in an amount sufficient to treat a particular disease state.

Description

EIGE EFFICIENCY EX ~7KO TRANSDUCTION OF
EEMATOPOIETIC STEM ~li'.~,T.~ BY RECOMBINANT
RETROVIRAL PREPARATIONS

~echnical Field The present invention relates generaDy to recomhin~nt retroviruses and gene therapy, and more specificaDy, to ~~co,.,hil~AI~l retroviral particle p,t;p~tions suitable for a variety of somatic cell gene therapy app1ic~tir~ne Bacl~ nd of the Invention Since the discovery of DNA in the 1940s and co"~ p through the most recent era of reco,..bi~.t DNA technology, s~ "I;~l lese~.,h has been undertaken in order to realize the possibility that the course of disease may be ~ Il'Pcled through illLe. a.,Lion with the nucleic acids of living or~nieme Most l ece.lLly, a wide variety of metho~e have been des~;,ibed for altering or ~ p genes of somatic tissue (a process sQmtotim~s ler~ d to as "somatic gene therapy"), in~ flin~ for ey~mple~ viral vectors derived from retroviruses, adenoviruses, vaccinia viruses, herpes viruses, and adeno-~eeo~ ted viruses (see Jo~y, Cancer Gene Therapy 1(1):51-64, 1994), as well as direct h~srt:r techniques such as lipofe~;l;on ~;elgner - et al., Proc. NatL Aca~ Sci. USA 84:7413-7417, 1989), direct DNA injection (Acsadi ef a~., Nature 352: 815-818, 1991), rnicroprojectile bo...ba. d...ent (Williams et al., PNAS 88:2726-2730, 1991), liposomes of several types (see, e.g, Wang et al., PNAS 84:7851-7855, 1987) and ~-lminietration of nucleic acids alone (WO 90/11092).
Ofthese te~hniqu~oe lc~co.. h;.. n~.l retroviral gene delivery methods have been most extensively utili7~ in part due to: (1) the effi~ierlt entry of genetic material (the vector genome) into replic~ting cells; (2) an active, Pffirient process of entry into the target cell mlcle~ls; (3) relatively high levels of gene t:Aples:iion; (4) the potential to target particular cellular subtypes through control of the vector-target cell binding and the tissue-specific 30 control of gene expression; (5) a general lack of pre-existing host immlmity; and (6) substantial knowledge and clinical experience which has been gained with such vectors Briefly, retroviruses are diploid positive-strand RNA viruses that replicate through an integrated DNA interm~ te In particular, upon infection by the RNA virus, the retroviral genome is reverse-transcribed into DNA by a virally encoded reverse transcriptase that is 35 carried as a protein in each retrovirus. The viral DNA is then integrated pseudo-randomly SUBSTITUTE SHEET (RULE 26) into the host cell gPnnrne ofthe ;..r~ ...p cell, folll",lg a "provirus" which is inherited by yhtPr cells.
Wlld-type retroviral P~o...es (and their proviral copies) contain three genes (the gag, pol and env genes), which are preceded by a pacl S~yi~ signal (yr), and two long tersninal S repeat ~LTR) seqU~nrpc which flank both ends. Briefly, the gag gene ~onrodes the internal ;.t. ..~,lu.~ (m~rlçoc~rs;(l) pruleins. Thepol gene codes for the RNA-dep~n~nt DNA
polymerase which reverse ll~ sclil,es the RNA g~o.~.e, and the env gene P!nrodes the r~lluvil~ll envelope gly~;opluleins. The 5' and 3' LTRs contain cis-acting e1~ompntc n~ce to p-ulllole l.~scli~lion and polyadenylation of retroviral RNA
~-ljacent to the 5' LTR are sequences nPcP~ for reverse ll~ scli~lion ofthe ~e~--,-- .ç (the tRNA primer binding site) and for effi~ ;ent el~r~l~C ~l~tic n of retroviral RNA into particles (the psi sequence). Removal of the p?~r~glnf~ signal prevents enr~r~ tion (pp~r~ ing of retroviral RNA into infectiollc virions) of g~nnmic RNA, ~ltholl~h the r~clllting mutant can still direct synthesis of all prot~ls Pncoded in the viral pel-n---P
~cs.. l ~il- ~ retroviruses and various uses thereof have been des-,lil,ed in mlll.crous ere cnces inr.ll~lin~, for PY~mplç, Mann, etal. (Cell 33:153, 1983), Cane and lunl~ n (Proc. Nat'L Acad. Sci. USA 81:6349, 1984), Miller, et al., Human Gene T7~erapy 1:5-14, 1990, U.S. Patent Nos. 4,405,712; 4,861,719; 4,980,289 and PCT Applic~tinn Nos. WO
89/02,468; WO 89/05,349 and WO 90/02,806). Briefly, a foreign gene of interest may be i--co- ~,o-~led into the retrovirus in place of a portion of the normal retroviral RNA. When the retrovirus injects its RNA into a cell, the foreign gene is also introduced into the cell, and may then be ~-leg ~td into the host's cellular DNA as if it were the retrovirus itself.
Expression of this foreign gene within the host results in e Ayres:iion of the foreign protein by the host cell.
One disadvantage, however, of .eco~lbilla"l retroviruses is that they p,in.il.ally infect only replicating cells, thereby making ~ffir;~nt direct gene transfer difficult or impossible for cells characterized as largely non-replir~tinP such as hematopoietic stem cells. Indeed, some scientictc have sug~sted that other, more f!ffiri~nt mPthsrlc of gene transfer, such as direct ~lminictration of pure plasmid DNA, be utilized ~avis et al., Human Gene Therapy 4:733-740, 1993) to introduce nucleic acid molec~ os into such cells.
In order to increase the efficacy of recombinant retroviruses, the methods which have been suggested have principally been aimed at infl~lring the desired target cells to replicate, thereby allowing the retroviruses to infect the cells. Such methods have inrlllderl for example chemical tre~tment with 10% carbon tetrachloride in mineral oil (Kaleko, et al., 3~ Human Gene Therapy 2:27-32, 1991). However, such techniques are not p,~rt:lled for use SUBSTITUTE SHEET lRULE 26) in ex vivo techniques cl~RcignRd to introduce nucleic acid molec~llec encoding thelape~lLic gene products into ~ n hematopoietic cells.
~mm~ n hematopoietic cells provide a diverse range of physiological activities.
These cells are divided into lyll~phoi~, myeloid and erythroid li~ gec The lymphoid - 5 linR~geS ~o~ g B-cells, T-cells, and NK-cells, provides for the produrtinn of ~ .o~ C, re~ tion ofthe cellular imm--ne system, dele~il;Q~ offoreign agents in t~reb~, *~v~
of cells foreign to the host, and the like. The myeloid lineage, which inrl~ldes monocytes, granulocytes, mR~k~ryocytes as well as other cells, monitors for the prêsel-ce of foreign bodies, provides pl ote~ilion against neoplastic cells, scavenges foreign materials, produces pl~ ; and the like. The erythroid lineage provides the red blood cells, which act as oxygen carriers.
Despite the diversity of the nature, morphology, characteristics and fimrtinn Ofh~ .-.atol)oi~ Lic cells, these cells are derived from a single hematopoietic p, ogel i~or cell poplll~tion termed ~Istem cells." Stem cells are capable of self-regeneration and may become lineage restricted progenitors, which further di~èlell~iaLe and expand into specific lin~e5 As used herein, the term 'Istem cells" or "helllalopoietic stem cells" refers to he~llaLopoieLic cells and not stem cells of other cell types. Further, unless in~lic~ted otherwise, "stem cells"
refers to human hellla~OpOietic stem cells. U.S. Patent No. 5,061,620 desc-il,es a s~ lly homo~ ollc stem cell composition and the manner of ol.~ 2 such a composition. See also the ~ere.cnces cited therein.
Stem cells cQnctihlte only a small pe.-,elllage ofthe total number of helllalop~ c cells. ~m~topoietic cells are i-l~ntifi~hle by the p,csencc of a variety of cell surface 'Imarkers." Such Ill~kel~ may be either specific to a particular lineage or progenitor cell or be present on more than one cell type. CD34 is a marker found on stem cells and a eignific~nt number of more di~el~lu~ed progenitor cells. U.S. Patent No. 4,714,680 describes a population of cells ~ ,l e~ing the CD34 marker.
Table 1 ~UIIII~ és probable phenotypes of stem cells in fetal, adult, and mobilized peripheral blood. In Table l, myelomonocytic stands for myelomonocytic ~ori~ted Ill~kel~, NK stands for natural killer cells, FBM and ABM refer to fetal and adult bone marrow, respectively, and AMPB stands for adult mobilized peripheral blood. As used herein both infra, supra and in Table 1, the negative sign or, uppercase negative sign, (-) means that the level ofthe specified marker is llnrietect~hle above Ig isotype controls by FACS analysis, and in~ des cells with very low e"~lession of the specified marker.
.

SU~STITUTE SHEET (~ULE 26) TABLE I

ble Slem Ccll Phcr NIC ~nd T~ll B~:U M~ e~ ~ ~ J' ~~
~e~s CD CD CD CDI CDI CD2 CDI CDI CDI CD3 CD3 CD3 HL~ D C ~

2 3 ~I 0 9~) 4 5 6 3 4 1~
FBM -- -- -- -- -- -- -- -- -- ~ + _ + + + io +
ABM -- -- -- -- ---- -- -- -- -- + + W- + + b +
AMPB -- -- -- -- -- -- -- -- -- W- + t W- + + b +

The ability of stem cells to undergo subst~nti~l self-renewal as well as the ability to proliferate and dirr~ l ~"LiaLe into all of the hematopoietic lineages makes stem cells the target of choice for a number of gene therapy applir,~tiQnc Succeccfi~l gene ll~nsrer into stem cells should provide long-term repopulation of an individual with the morlified cells and their progeny, which will express the desired gene product. By contrast, gene h~ rel into more mature hematopoietic cells, such as T-cells, at best, provides only transient therapeutic benefit. Thus, there have been world-wide efforts toward finding effective methods of g~n~tir~lly modifying stem cells. For reviews of genetic msrlifir~tion of stem cells see Brenner (1993) J. ~em~tother. 2:7-17; Miller (1992) Nature 357:455- 160; and Nlenhuis (1991) Cancer 67:2700-2704.
Most efforts to g~n~tic~lly modify stem cells have involved the use of retroviral vectors. Other methods such as liposome-m~ ted gene transfer or adeno-~csori~ted viral vectors have also been used.- As diccllcced previously, retroviral vectors have been the primary vehicle due to the generally high rate of gene transfer obtained in t;A~.Jtl hllents with cell lines, and the ability to obtain stable integration of the genetic material, which ensures that the progeny ofthe modified cell will contain the transferred genetic material.
Efficient gene transfer into human stem cells has proven difficult due to a variety of factors. Currently used methods of retroviral tr~ncd~lction into human stem cells have a number of practical limis~tiorlc One limitation is the extremely low numbers of stem cells present in any tissue. Therefore, in tr~ncd~ctions performed with relatively impure SUBSTITUTE SHEET (RULE 26) Most efforts to g~netic~lly modify stem cells have involved the use of retroviral vectors. Other methods such as liposome-mtocli~ted gene transfer or adeno-associated viral vectors have also been used. As discussed previously, retroviral vectors have been the primary vehicle due to the generally high rate of gene transfer obtained in experiments with - 5 cell lines, and the ability to obtain stable integration of the genetic material, which ensures that the progeny of the modified cell wil} cont~n the k all.,r~ ;d genetTc IllaLc;lial.
Efficient gene transfer into human stem cells has proven difficult due to a variety of factors. Currently used methods of retroviral transduction into human stem cells have a number of practical limitations. One limitation is the extremely low numbers of stem cells present in any tissue. Therefore, in tr~n.~d~-ctions performed with relatively impure populations of cells, the ratio of virus particles to stem cells will be quite low. This limitation is compounded by the relatively low titers generally obtained with most retroviral vectors, typically in the range of 105 to 1 o6 infectious virions per millilit~r. Also, the effect of more difre~ ted cells in culture on the growth or division of stem cells is not well understood.
In addition, primitive stem cells typically are quiescent in culture; retroviral vectors require target cells to be cycling for stable integration of the retroviral DNA. Cytokines have been used to cause stem cells to cycle, which improves gene transfer efficiency, but the effect of various cytokines in driving stem cells to dirrel ell~iation remains in question.
The range of host cells that may be infected by a retrovirus or retroviral vector is deter nined by the viral envelope protein. Therefore, a lack or deficiency of the receptor for the given envelope protein would limit transduction efficiency. In addition, a lack of the requisite cellular factors involved in viral binding, penetration, uncoating of the retroviral vectors, viral replication or integration would limit tr~n~ c.tion efficiency.
It is the object of the present invention to provide ex vivo methods for using compositions of recolllbinall~ retroviral particles to deliver vector constructs encoding genes of interest to hematopoietic stem cells ex vivo. The tr~ncd~lced stem cells may then be re-minictered to the patient by standard techniques, e.g, intravenous infusion to achieve a desired therapeutic benefit.

Summar~ ofthe Invention ~ The present invention provides compositions and methods for transducing hematopoetic stem cells. Within one aspect of the invention a method is provided for the production of transduced hematopoetic stem cells comprising obtaining a population of

3 ~ hematopoetic stem cells from a patient and transducing the population of hematopoetic stem SUBSTmJTE SH EET tRULE 2~i) cells with a recombinant retroviral particles substantially free from co.,~ tion with replication competent retrovirus, wherein the reco~ hl~llL retroviral particles carry a vector construct encoding a gene of interest. Within one embodiment of the invention wherein the vector construct encodes a molecule selected from the group consisting of a protein, an 5 active portion of a protein and a RNA molecule with intrinsic biological activity. The protein or ~ctive portion of a protein is selected from the group con~i~ting of a cytokine, a colony stim~ tinf~ factor, a clotting factor, and a hormone. Within another embodiment of the invention methods are provided for treating a genetic disease, cancer, infectious disease, degenerative disease, infl~mm~tory disease, cardiovascular disease, and autoi.. ll-e disease 10 by ~rlmini.ct~ring to a patient a composition or reintroduction of a therapeutically effective amount ofthe population oftr~n~d~-ced hematopoetic stem cells. In another embodiment the tr~ned~lced population of hematopoetic stem cells are characteri7~d as CD34+Thy- 1+Lin~.
In another embodiment the reccm.l)i,lal,L reroviral vectors used to the tr~n.~ -ce the hematopoitic stem cells are xenotropic retroviral vectors. Pl~ Lbly the rerotroviral vector 15 ~,~a,~lions used to tr~n.~dtlce the hematopoietic stem cells are high titer plc:l)a~ions. In yet another embodiment the hematopoetic stem cells are eYr~nf~ed in vitro prior to re-introduction of the cells into the patient.
In another aspect of the invention an in vivo delivery vehicle comprising transplantable hematopoetic stem cells which express a therapeutically effective amount of a 20 gene product encoded by a gene wherein the gene does not occur in hematopoetic stem cells or where the gene occurs in hematopoetic stem cells but is not expressed in the cells and wherein the gene can be modified to be expressed in hematopoetic stem cells is provided.
In other aspects of the invention the tr~n.~d~lced hematopoetic stem cells and compositions of hematopoetic stem cells encoding a gene of interest, hematopoetic stem cells 25 tr~ncd~lced with a reco",bi,.al,l xenotropic retroviral vector, and hematopoetic stem cells char~ctt-ri7~d as CD34+Thy-1+Lin~ tr~n~d~-c.ed with recolllbillallL retroviral particles are provided. In one embodiment of this aspect of the invention compositions are provided substantially free from co..l~...il-~l;on with replication competent retrovirus.

Definition of Terms The following terms are used throughout the specification. Unless otherwise indicated, these terms are defined as follows:

SUBST~TUTE SHEET (RULE 26) "Event-specific promoter" refers to transcriptional promoter/~nh~ncçr or locus d~fining elements, or other elements which control gene expression as tli~c~ssed above, whose transcriptional activity is altered upon response to cellular stimuli. Representative examples of such event-specific promoters include thymidine kinase or thymidylate syntheses - 5 promoters, alpha or beta i~lLelrt;,oll promoters and promoters that respond to the presence of hormones (either natural, synthetic or from other non-host o~
"Tissue-specific promoter" refers to transcriptional promoter/çnh~n~çr or locus d~fining elements, or other elements which control gene cA~l~;s~,ion as discussed above, which are pl c:rel ~lLially active in a limited number of hematopoietic tissue types.
10 Representative examples of such hematopoietic tissue-specific promoters include, but are not limited to, the IgG promoter, a- or ~-globin promoters, T-cell receptor promoter, Cl~y---e A, Gl ~y...e B, CD8, and CD 1 lb.
"Tr~n.~d~lctinn" involves the association of a replication defective, reco---bi--~lL
retroviral particle with a cellular receptor, followed by introduction of the nucleic acids 15 carried by the particle into the cell. "Transfection" refers to a method of physical gene Ll ~ ,r~. wherein no I c:LI ùvi~ ~I particle is employed.
"Vector construct", "I~Liuvi-~I vector", ~leco~"bina"L vector", and "reco--,binanL
retroviral vector" refer to a nucleic acid construct capable of dil e~iLi--g the CA~Jl ession of a gene of interest. The retroviral vector must include at least one transcriptional 20 promoter/enhancer or locus d~fining element(s), or other ~l~m~n~.~ which control gene t~A~I ~ssion by other means such as alternate splicing, nuclear RNA export, post-translational modification of mç~ ng~r, or post-transcriptional modification of protein. Such vector constructs must also include a p~ ging signal, long t~rmin~l repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites app-op-iate to the retrovirus 25 used (if these are not already present in the retroviral vector). Optionally, the vector construct may also include a signal which directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way example, such vectors will typically include a 5' LTR, a tRNA binding site, a p~.k~rng signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof. In order to express a desired gene 30 product from such a vector, a gene of interest encoding the desired gene product is also included.
.

Numerous aspects and advantages of the invention will be apparent to those skilled in ~ the art upon consideration of the following detailed description which provides illumination 3 5 ofthe practice ofthe invention.

SUBSTITUTE SHEET (RULE 26) Detailed Description of the Invention The present invention is based on the unexpected discovery that recol,.bi,.~.L
5 retroviral particles callying a vector construct cnmpri.~ing a gene of interest can be used ex to efficiently tr~n~ ce h~ ..a~o~uoietic stem cells. As a result, l~col..bi--allL retroviral partides accc,l ~ling to the invention can be used for purposes of somatic gene therapy. A
more thorough description of such 1 ~;collllJill~ll~ retroviral particles, their production and p~c~ging and uses therefore is provided below.
Generation of Reco~bi.~anL Retroviral Vectors As noted above, the present invention provides compositions and methods comprising reco...l~ a~ -uvil~l particles, inr.lll-linp recolll~ill~-~ xenotropic retroviral 15 vector particles, for use in ex vivo somatic gene therapy. The construction of reco---l,ind-~L
~Lluvil~l vectors and particles is described in greater detail in an application entitled "Recon.l,il.a..L ReLluvil~lses". Production oftr~n~ ctinn co---p~;~el-~ leco---l,i--d -L xenotropic retroviral particles is described in U.S.S.N. 08/156,789, and U.S.S.N. 07/965,084, which are hereby incorporated by reference in their entirety. In general, the l ecol--bil-~-L vector 20 constructs described herein are p-c:pa~d by selecting a plasmid with a strong promoter, and ~p.up.i~Le restriction sites for insertion of DNA sequences of interest downstream from the promoter.
According to the invention, the recol~ ..L vector construct is carried by a recombinant retrovirus. Retroviruses are RNA viruses with a single positive strand genome 25 which in general, are nonlytic. Upon infection, the retrovirus reverse transcribes its RNA
into DNA, forming a provirus which is inserted into the host cell genome. The retroviral genome can be divided conceptually into two parts. The "trans-acting" portion consists of the region coding for viral structural proteins, incl~lrling the group specific antigen (gag) gene for synthesis of the core coat proteins; the pol gene for the synthesis of the reverse 30 transcriptase and integrase enzyrnes; and the envelope (env) gene for the synthesis of envelope glycoproteins. The "cis-acting" portion consists of regions of the genome that is finally packaged into the viral particle. These regions include the pa~.k~ging signal, long temlinal repeats (LTR) with promoters and polyadenylation sites, and two start sites for DNA replication. The intemal or "trans-acting" part of the cloned provirus is replaced by the , ~ gene of interest to create a "vector construct". When the vector construct is placed into a SUBSTITUTE SHEET (RULE 26) _ g _ cell where viral p~r.l~gin~ proteins are present, the transcribed RNA will be p~rl~gçd as a viral particle which, in turn, will bud offfrom the cell. These particles are used to tr~n.cd-lce tissue cells, allowing the vector construct to integrate into the cell genome. Although the vector construct expresses its gene product, the virus carrying it is replication defective - 5 because the trans-acting portion of the viral genome is absent. Various assays may be uti~zed in o~der to detect the presence ~Jf any i ~ ,aLiu~ ;IIL ;I~,Liuu., 1 ~ Vil ~IS.
One p-~r~lled assay is the Pxtto.nrled S+L- assay described in Example 4.
In the broadest terms, the retroviral vectors of the invention comprise a transcriptional promoter/~nh~ns~r or locus d~fining ~l~m~nt(s)~ or other ~l~m~ntc which 10 control gene t;A~I ession by other means such as ~ltprn~te splicing, nuclear RNA export, post-translational modification of mPss~nger, or post-transcriptional modification of protein.
Such vector constructs must also include a p~qr~ging signal, long tf!rmin~l repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropliate to the retrovirus used (if these are not already present in the retroviral vector). Optionally, the 15 vector construct may also include a signal which directs polyadenylation, as well as one or more restriction sites and a translation telll,illalion sequence. By way example, such vectors will typically include a 5' LTR, a tRNA binding site, a p~c~ ing signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof. Such vectors do not contain one or more of a complete gag,pol, or env gene, thereby r~n-l~ring them replication 20 incompetent. In addition, nucleic acid molecules coding for a selectable marker are neither required nor pl ert~ ;d.
Preferred retroviral vectors contain a portion of the gag coding sequence, preferably that portion which comprises a splice donor and splice acceptor site, the splice acceptor site being positioned such that it is located ~dj~clont to and u~sLI ~alll from the gene of interest. In 25 a particularly pr~rel I ed embodiment, the gag transcriptional promoter is positioned such that an RNA transcript initi~ted therefrom contains the 5' gag LTR and the gene of interest. As an alternative to the gag promoter to control CA~ s~ion of the gene of interest, other suitable promoters, some of which are described below, may be employed. In addition, alternate enhancers may be employed in order to increase the level of expression of the gene ~0 of interest.
In pl ~r~. I ed embodiments of the invention, retroviral vectors are employed, particularly those based on Moloney murine leukemia virus (MoMLV). MoMLV is a murine retrovirus which has poor infectivity outside of mouse cells. The related amphotropic N2 retrovirus will infect cells from human, mouse and other org~ni.cmc. Other preferred 5 retroviruses which may be used in the practice of the present invention include gibbon ape SUBSTITUTE SHEET (RULE 26) WO 96/33281 PCI'IUS96/05432 le~lk~ virus (GALV) (Todaro, etal, Virology, 67:335, 1975; Wilson, etal, J. Vir., 63:2374, 1989), feline immllnodeficiency virus (FIV) (Talbatt, ef al, Proc. Nat'l. Acad. Sci.
USA, 86:5743, 1984), and feline lellk~ virus (FeLV) (Leprevette, etal, J. Vir, 50:884, 1984; Elder, etal., J. Vir., 46:871, 1983; Steward, etaL, J. Vir., 58:825, 1986; Riedel, et al., J. Vir., 60:242, 1986), although retroviral vectors according to the invention derived fr~nn other type C retroviruses (Weiss, RATA Tumor ~irases, vols. I and II, Co~d Spring Harbor Laboratory Press, N.Y.) can also be generated.
A variety of promoters can be used in the vector constructs of the invention, inrlll-1inf~ but not n~cçc~rily limited to the cytomegalovirus major imml~di~te early promoter (C:~MV MIE), the early and late SV40 promoters, the adenovirus major late promoter, thymidine kinase or thymidylate synthase promoters, alpha or beta ill~elreloll promoters, event or tissue specific promoters, etc Promoters may be chosen so as to potently drive high levels of expression or to produce relatively weak ~ e~sion, as desired. As those in the art will appl eciaLe, numerous RNA polymerase II and RNA polymerase III dependent promoters can be utilized in pr~cti~in~ the invention.
In one embodiment, reccj,lll~illall~ rt:~l uvil ~l vectors comprising a gene of interest are under the transcriptional control of an event-specific plolllo~er, such that upon activation of the event-specific promoter the gene is expressed. Numerous event-specific promoters may be utilized within the context ofthe present invention, inrlllrlin~ for example, promoters which are activated by cellular proliferation (or are otherwise cell-cycle dependent) such as the thymidine kinase or thymidylate synthase promoters (Merrill, Proc. Natl. Acad. Sci. USA, 86:4987, 1989; Deng, et al., Mol. Cell. Biol., 9:4079, 1989); or the ~ r~llill receptor promoter, which will be ~l~nsclil)~ionally active primarily in rapidly proliferating cells (such as hematopoietic cells) which contain factors capable of activating transcription from these promoters pl t; rel t;ll~ially to express gene products from gene of interest; promoters such as the alpha or beta hl~elrelon promoters which are activated when a cell is infected by a virus (Fan and Maniatis, EMBO J., 8:101, 1989; Goodbourn, etal., Cell, 45:601, 1986); and promoters which are activated by the presence of hormones, e.g., estrogen response promoters. See Toohey et al., Mol. Cell. Biol., 6:4526, 1986; and promoters that are activated in response to cellualr stress or insult, e.g., electrophilic response elements (Friling.
et al., PNAS, 8~:6258, 1990) In another embodiment, recombinant retroviral vectors are provided which comprise a gene of interest under the transcriptional control of a tissue-specific promoter, such that upon activation of the tissue-specific promoter the gene is expressed. A wide variety of 3 ~ tissue-specific promoters may be utilized within the context of the present invention SUBSTITUTE S~EET (RULE 26) WO 96/33281 PCTtUS96/05432 Representative examples of such promoters include: B-cell specific promoters such as the IgG promoter; T-cell specific promoters such as the T-cell receptor promoter (Anderson, et al., Proc. Nafl. Acad. Sci. USA, 85:3551, 1988; Winoto and R~ltimore, EMZ30 J., 8:29, 1989); bone-specific promoters such as the osteocalcin promoter (Markose, ef aL, Proc.
Nafl. Acad. Sci. USA, 87: 1701, 1990; McDonnell, ef al., Mol. Cell. Biol, 9:3517, 1989;
Kerner, et al., Proc. Nafl. Acad. Sci. USA, 86:4455, 1~89), the E,-2 Itl~tlllUt~l, E,-2 lc~
promoter, and the MHC Class II promoter, and hematopoietic tissue specific promoters, for in.ct~n-~e erythoid specific-transcription promoters which are active in erythroid cells, such as the porphobilinogen de~min~e promoter (Mignotte, ef al., Proc. Nafl. Acad. Sci. USA, 10 86:6458, 1990), a- or 13-globin specific promoters (van ~sen~lçl~ et al., Cell, ~6:969, 1989, Forrester, etal., Proc. Natl. Acad. Sci. USA, 86:5439, 1989), endothelial cell specific promoters such as the vWf promoter, mçg~k~ryocyte specific promoters such as ~-thromboglobulin, and many other tissue-specific promoters. Examples of promoters that may be used to cause ~A~ s~ion of the introduced sequence in specific cell types include 15 Granzyme A and ~, ~yl~le B for expression in T-cells and NK cells, the CD34 promoter for eA~l~s~ion in stem and progenitor cells, the CD8 promoter for expression in cytotoxic T-cells, and the CDl lb promoter for eA~Jl es~ion in myeloid cells.
Retroviral vectors according to the invention may also contain a non-LTR ~nh~n~er or promoter, e.g, a CMV or SV40 çnh~n~ .r operably associated with other elements 20 employed to regulate CA~]I es~ion of the gene of interest. Additionally, retroviral vectors from which the 3' LTR çnh~n~çr has been deleted, thereby inactivating the 5' LTR uponintegration into a host cell genome, are also contemplated by the invention. A variety of other elements which control gene eA~tl es~ion may also be utilized within the context of the present invention, including, for example, locus-cl~.fining elements such as those from the 25 ~-globin gene and CD2, a T-cell marker. In addition, elements which control expression at the level of splicing, nuclear export, and/or translation may also be in~lllded in the retroviral vectors. Representative examples include the ~-globin intron sequences, the rev and rre elements from HIV-l, the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV), a 219 nucleotide sequence that allows rev-independent replication of rev-30 negative HIV proviral clones, and a Kozak sequence. Rev protein functions to allow nuclear export of unspliced and singly spliced HIV RNA molecules. The MPMV element allows nuclear export of intron-cont~ining mRNA. The CTE element maps to MPMV nucleotides 8,022-8,240 (Bray, et al., Biochemist7y, 91: 1256, 1994).
In another plere"~d embodiment, the retroviral vector contains a splice donor (SD) 35 site and a splice acceptor (SA) site~ wherein the SA is located upstream ofthe site where the SUBS~lTUTE SHEET (RULE 26) gene of interest is inserted into the recombinant retroviral vector. In a plerell~d embodiment, the SD and SA sites will be separated by a short, i.e., less than 400 nucleotide, intron sequence. Such sequences may serve to stabilize RNA transcripts. Such stabilizing sequences typically comprise a SD-intron-SA configuration located 5' to the gene of interest.
The recombinant retroviral vectors of the invention will also preferably contain~a.lscli~Liollal promoters derived from the ~ ;ull u~,.~ly p~ iùl~ srrch that a resultant transcript COlllpliSillg the gene of interest further comprises a 5' gag LTR
(unL~ ted region) u~usl,ea,,, ofthe gene of interest.
The present invention also provides for multivalent vector constructs, the 10 construction of which may require two promoters when two proteins are being e:~l essed, because one promoter may not ensure adequate levels of gene ~ ssion of the second gene.
In particular, where the vector construct expresses an ~nti.c~n~e message or ribozy-me, a second promoter may not be n~c~ss~ry Within certain embo(iim~nt~, an internal ribosome binding site (IRBS) or herpes simplex virus thymidine kinase (HSVTK) promoter is placed in 15 conjunction with the second gene of interest in order to boost the levels of gene expression of the second gene. Briefly, with respect to IRBS, the ul,s~ ", ullLl ,.n~l~ted region of the immlmnglobulin heavy chain binding protein has been shown to support the internal engagement of a bicistronic message (Jacejak, et al., Nature 353:90, 1991). This sequence is small, applc,xillla~ely 300 base pairs, and may readily be incol~ol~led into a vector in order 20 to express multiple genes from a multi-cistronic m~c~e whose cistrons begin with this sequence.
Retroviral vector constructs accordil,g to the invention will often be encoded on a plasmid, a nucleic acid molecule capable of propagation, segregation, and extra-chromosomal m~inten~nçe upon introduction into a host cell. As those in the art will 25 understand, any of a wide range of existing or new plasmids can be used in the practice of the invention. Such pl~cmi~lc contain an origin of replication and typically are modified to contain a one or more multiple cloning sites to f~ t~te rec~llbil,a"L use. Preferably, plasmids used in accordance with the present invention will be capable of propagation in both eukaryotic and prokaryotic host cells.

SUBSTITUTE SHEET (RULE 26) Generation of P~k~in~ Cells Another aspect of the invention relates to methods of producing recombinant retroviral particles incorporating the retroviral vectors described herein. In one embodiment, - 5 vectors are packaged into infectious virions through the use of a pacL-~ing cell. Briefly, a F~r.k~ging cell is a cell comprising, in additiorr t~ its n~l gerretrc e~ Jh.~ L, ~hl;L;ulla nucleic acids coding for those retroviral structural polypeptides required to package a retroviral genome, be it recombinant (i. e., a retroviral vector) or otherwise. The retroviral particles are made in p~ ing cells by COlll~illillg the leLlovil~LI genome with a capsid and envelope to make a tr~n.e~ ction competent, preferably rep1ic~tion defective, virion. Briefly, these and other pac~ in~ cells will contain one, and preferably two or more nucleic acid molecules coding for the various polypeptides, e.g, gag, pol, and env, required to package a retroviral vector into an infectious virion. Upon introduction of a nucleic acid molecule coding for the retroviral vector, the p~ gin~ cells will produce infectious leLIOvili~l particles. Pack~ing cell lines transfected with a retroviral vector according to the invention which produce infectious virions are lerelled to as "producer" cell lines.
A wide variety of animal cells may be utilized to prepare the p~ gin~ cells of the present invention, int~ r1ing without limitation, epithelial cells, fibroblasts, hepatocytes, endothelial cells, myoblasts, astrocytes, Iymphocytes, etc.. PlerèlêllLially~ cell lines are selected that lack genomic sequences which are homologous to the retroviral vector construct, gag/pol expression c~e~ette and env eAlJlès:iion ~ eSette to be utilized Methods for determining homology may be readily accompli~h~d by, for example, hybridization analysis (Martin et al., Proc. Na~l. Acad. Sci., USA, 78:4892, 1981; and U.S.S.N.
07/800,921, supra) The most common p~c.k~ging cell lines (PCLs) used for MoMLV vector systems (psi2, PA12, PA317) are derived from murine cell lines. However, murine cell lines are typically not the pleÇelled choice to produce retroviral vectors intt~nrled for human therapeutic use because such cell lines are known to: contain endogenous retroviruses, some of which are closely related in sequence and retroviral type to the MLV vector system prefel ~ ed for use in practicing the present invention; contain non-retroviral or defective retroviral sequences that are kno~,vn to package efficiently; and cause deleterious effects due to the presence of murine cell membrane components.
An important consideration in developing p?~ ging cell lines useful in the invention is the production therefrom of replication incompetent virions, or avoidance of generating 3~ replication-competentretrovirus(RCR)(Munchau, etal. I~irolog~ 176:26~. 1991) This SUBSTITUTE SHEET (RULE 26) will ensure that infectious retroviral particles harboring the recolllbinall~ retroviral vectors of the invention will be incapable of independent replication in target cells, be they in vitro or in vivo. Independent replication, should it occur, may lead to the production of wild-type virus, which in turn could lead to multiple integrations into the chromosome(s) of a patient's cells, thereby increasing the possibility of insertional mutagenesis and its associated problems.
l~R production can occur in at least two ways~ ~rough ~rcnnologous recomlJi~ld~
between the therapeutic proviral DNA and the DNA encoding the retroviral structural genes ("gag/pol" and "env") present in the pAc lrAging cell line; and (2) generation of replication-competent virus by homologous recolllbillalion of the proviral DNA with the very large 10 number of defective endogenous proviruses found in murine pA~lrAging cell lines.
To circumvent inherent safety problems associated with the use of murine based eCOIll~ill~ll retroviruses, as are prerelled in the practice ofthis invention, pAçL Aging cell lines may be derived from various non-murine cell lines. These include cell lines from various ~ llllllAie, in~ linf~ hllmAn.~, dogs, monkeys, mink, hallls~el~, and rats. As those in 15 the art will appreciate, a mllhit~lde of paC:~Aging cell lines can be generated using techniques known in the art (for in~tAn-~.e, see U.S.S.N. 08/156,789 and U.S.S.N. 08/136,739). In ,orerelled embo-limPnt~, cell lines are derived from canine or human cell lines, which are known to lack genomic sequences homologous to that of MoMLV by hybridization analysis (Martin el al., supra) A particularly plerelled parent dog cell line is D17 (ATCC. CRL
20 8543). HT-1080 (ATCC. CCL 121; Graham ef al., Vir., 52:456, 1973) and 293 cells (Felgner e~ al., Proc. Na~'l. Acad. Sci. USA 84:7413, 1987) leplesen~ particularly plerelled parental human cell lines. Construction of pa~'~Agillg cell lines from these cell lines for use in conjunction with a MoMLV based recolllb;llall~ retroviral vector is described in detail in U.S.S.N. 08tl56,789, supra Thus, a desirable prerequisite for the use of retroviruses in gene therapy is the availability of retroviral p~c~ging cell lines incapable of producing replication competent, or "wild-type," virus. As pacL-~ging cell lines contain one or more nucleic acid molecules coding for the structural proteins required to assemble the retroviral vector into infectious retroviral particles, recombination events between these various constructs might produce 30 replication competent virus, i.e., infectious retroviral particles cont~inin~; a genome encoding all ofthe structural genes and regulatory elements, inçlllfling a pa~k~ging signal, required for independent replication. In the past several years, many di~el ell~ constructions have been developed in an attempt to obviate this concern. Such constructions include: deletions in the ,' LTR and portions of the 5' LTR (see, Miller and Buttimore, Mol. Cell. Biol, 6: '895, ~5 1986). where two recombination events are necessary to form RCR; use of complementar~

SVBSTITUTE SHEET (RULE 26) portions of helper virus, divided among two separate pl~cmicl~, one co~ ,;"p gag and pol, and the other coll~ ;llg env (see, Markowitz et al., J. ViroL, 62:1120, and Markowitz et al., Virology, I67: 600, 1988), where three reconlbillà~ion events are required to generate RCR.
The ability to express gag/pol and env function separately allows for manipulation of these functions independently. A cell line that expresses ample amounts of gag/pol can be used, for example, to address questions of titer w~r regd~ to e~ Vll~, laeL~ ~ iTr measured low titers is the density of appl opliate receptor molecules on the target cell or tissue. A second factor is the affinity of the receptor ror the retroviral envelope protein.
One report suggests that xenotropic vector, in the presence of replication-complement virus, 10 may more effectively infect human hematopoietic progenitor cells (Eglitis, et al., Biochem.
Biophys. Res. Comm. 151:201, 1988). vector-co"l~i";"~ particles, in the presence of replication-competent xenoL,~ic virus, also infect cells from other species which are not easily infectable by amphotropic virus such as bovine, porcine, and equine cells (Delouis, et al., Biochem. BiophysRes. Comm. 169:80, 1990). In a plert,ll~d embodiment ofthe 15 invention, p~c~ging cell lines which express a xenotropic env gene are provided.
Si nific~ntly, recolnbilldllL xenotropic retroviral particles produced from such pa(cl~ging cell lines are substantially free from association with replication competent retrovirus ("RCR").
More recently, further improved methods and compositions for inhibiting the production of replication incompetent retrovirus have been developed. See co-owned 20 U.S.S.N. 09/028,126, f~led September 7, 1994. Briefly, the spread of replication competent retrovirus generated through recolllbilld~ion events between the recolllbinallL retroviral vector and one or more of the nucleic acid constructs coding for the retroviral structural proteins may be prevented by providing vectors which encode a non-biologically active inhibitory molecule, but which produce a nucleic acid molecule encoding a biologically active inhibitory 25 molecule in the event of such recombination. The ~ s:,ion of the inhibitory molecule prevents production of RCR either by killing the producer cell(s) in which that event occurred or by suppressing production of the retroviral vectors therein. A variety of inhibitory molecules may be used, inclutlin~ ribozymes, which cleave the RNA transcript of the replication competent virus, or a toxin such as ricin A, tetanus, or diphtheria toxin, 30 herpes thymidine kinase, etc. As those in the art will appreciate, the te7~.hing~ therein may be readily adapted to the present invention.
In addition to issues of safety, the choice of host cell line for the packaging cell line is of importance because many of the biological properties (such as titer) and physical properties (such as stability) of retroviral particles are dictated by the properties of the host 35 cell For instance, the host cell must efficiently express (transcribe) the vector RNA _enome.

SUBSTITUTE SHEET (RULE 26) -CA 022l6868 l997- lO- l7 prime the vector for first strand synthesis with a cellular tRNA, tolerate and covalently modify the MLV structural proteins (proteolysis, glycosylation, myristylation, and phosphorylation), and enable virion budding from the cell membrane. For ex~mple7 it has been found that vector made from the mouse pa~ gin~ line PA317 is retained by a 0.3 5 micron filter, while that made from a CA line will pass through. Furthermore, sera from primates, inrlll~lin~ hllm~n.c, but not that from a wide variety of lower m~mm~l.c or birds, is known to inactivate ~ uvil-lses by an antibody independent complement lysis method. Such activity is non-selective for a variety of distantly related retroviruses. Retroviruses of avian, murine (in~ in~ MoMLV), feline, and simian origin are inactivated and Iysed by normal human serum. See Welsh, et al., Nature, 257:612, 1975; Welsh, et aL, Virology, 74:432, 1976; Banapour, et aL, Virology, 152:268, 1986; and Cooper, et al., (1986) Immunology of the Complement System, Pub. American Press, Inc., pp: 139-162. In addition, replication competent murine amphotropic l~LIuviluses injected i"Ll~Lv~;nously into primates in vivo are cleared within 15 minlltçs by a process m~ tecl in whole or in part by primate complement (Cornetta, et aL, Human Gene Therapy, 1: 15, 1990; Cornetta, et al., Human Gene Therapy, 2:5, 1991). However, it has recently been discovered that retroviral rçcict~nre to complement inactivation by human serum is mç~ te~, at least in some inct~n~.oc, by the p~clr~ ing cell line from which the retroviral particles were produced. Retroviruses produced from various human ~ac~ ing cell lines were ,~ L to inactivation by a component of human serurn, presumably complement, but were sensitive to serum from baboons and m~c~luec See colllmollly owned U.S.S.N. 08/367,071, filed on December 30, 1994. Thus, in a pl t:~ll ed embodiment of the invention, rec~, .,l ,;"~ retroviral particles are produced in human pac~ging cell lines, with parL-~ging cell lines derived from H~1080 or 293 cells being particularly plere,l~d.
In addition to generating infectious, replication defective recolllbillall~ retroviruses as described above, at least two other alternative systems can be used to produce recolllbillalll retroviruses carrying the vector construct. One such system (Webb, et al., BBRC, 190:536, 1993) employs the insect virus, baculovirus, while the other takes advantage of the m~mm~ n viruses vaccinia and adenovirus (Pavirani, et al., BBRC, 1~5:234, 1987). Each of these systems can make large amounts of any given protein for which the gene has been cloned. For example, see Smith, et al. (Mol. Cell. Biol., 3:12, 1983); Piccini, e~ al. (Meth.
En~vmolog~,~, 153:5~5, 1987); and Mansour el al. (Proc. Nafl. Acad. Sci. USA, 8 ~: 1359, 1985) These retroviral vectors can be used to produce proteins in tissue culture cells by insertion of appl Opl ;ate genes and, hence, could be adapted to make retroviral vector 3 ~ particles from tissue culture. In an adenovirus system, genes can be inserted into vectors and SUBSTITUTE SHEE~ (RULE 26) used to express proteins in m~mm~ n cells either by in vitro construction (Ballay, et al., ~:3861, 1985) or by recombination in cells (Thllmm~l, et al., J. Mol. Appl. Genetics, 1:435, 1982).
An alternative approach involves cell-free ps~cl~ging systems. For instance, retroviral 5 structural proteins can be made in a baculovirus system (or other protein production systems, such as yeast or E. coli) in a similar manner as described in Smith et al. (supra).
Recombinant retroviral genomes are made by in vitro RNA synthesis (see, for example, Flamant and Sorge, J. Virol., 62:1827, 1988). The structural proteins and RNA genomes are then mixed with tRNA, followed by the addition of liposomes with embedded env protein 10 and cell extracts (typically from mouse cells) or purified components (which provide env and other nece~s~ry processing, and any or other n~cess~. y cell-derived functions). The mixture is then treated (e.g, by sonication, temperature manipulation, or rotary dialysis) to allow ~nC~r~ tion of nascent retroviral particles. This procedure allows production of high titer, replication incompetent recc~ i"~,L retroviruses without co..~ ;on with pathogenic 15 retroviruses or replication-competent retroviruses.
Another important factor to consider in the selection of a p~rlr~ging cell line is the viral titer produced therefrom following introduction of a nucleic acid molecule from which the retroviral vector is produced. Many factors can limit viral titer. One of the most significant limiting factors is the ~, t;s~ion level of the pa~ ging proteins gag, pol, and env 20 In the case of retroviral particles, C~IJI t;s~ion of retroviral vector RNA from the provirus can also significantly limit titer. In order to select pac~ in~ cells and the resultant producer cells expressing high levels ofthe required products, an app~up~iate titering assay is required.
As described in greater detail below, a suitable PCR-based titering assay can be utilized.
In addition to preparing p~c~ging and producer cell lines which supply proteins for 25 par~gin~ that are homologous for the backbone of the viral vector, e.g, retroviral gag, pol, and env proteins for p~ctr~ging of a retroviral vector, pa~L ~gin~ and producer systems which result in chimeric viral particles, for in~t~nce a MoMLV-based retroviral vector packaged in a DNA virus capsid, may also be employed. Many other pac~ging and producer systems based on viruses unrelated to that of the viral vector can also be utilized, as those in the art 30 will appreciate.

Alterin~ the Host Ran~e of Recombinant Retroviral Particles Another aspect of the invention concerns recombinant xenotropic retroviral particles 3 ~ which have an altered host range as compared to retroviral particles cont~ining amphotropic SUBSmUTE S~EE~ tRULE 26~

envelope proteins. The host cell range specificity of a retrovirus is determined in part by the env gene products present in the lipid envelope. I~ ,Lingly, envelope proteins from one retrovirus can often substitute, to varying degrees, for that of another retrovirus, thereby altering host range of the resultant vector. Thus, p~ ~ng cell lines (PCLs) have been 5 generated to express either amphotropic, ecotropic, xenotropic, polytropic, or other c.lv~lo~e tropisms. Additionally, le~uv~uses accolding to the illv~llLi~ll which c~ntain "hybrid" or "chimeric" xenotropic envelope proteins can be similarly generated. Retroviral particles produced from any of these pacl~ging cell lines can be used to infect any cell which contains the corresponding distinct receptor (Rein and Schult_, Virology, 136:144, 1984).
The assembly of retroviruses is charact~n7ecl by selective inclusion of the retroviral genome and accessory proteins into a budding retroviral particle. IIlLele~,Lillgly, envelope proteins from non-murine retrovirus sources can be used for pseudotyping (i.e., the encapsidation of viral RNA from one species by viral proteins of another species) a vector to alter its host range. Because a piece of cell membrane buds offto form the retroviral 15 envelope, molecules normally in the Illellll,l~e may be carried along on the viral envelope.
Thus, a number of di~elellL potential ligands can be put on the surface of retroviral particles by manipulating the p~ gin~ cell line in which the vectors are produced or by choosing various types of cell lines with particular surface Ill~lh~
Briefly, in this aspect the present invention provides for enveloped l~:llovil~l particles 20 comprising: a nucleocapsid inr.ll-l1ing nucleocapsid protein having an origin from a first virus, which is a retrovirus; a p~c~g~kle nucleic acid molecule ~n~ orlin~ a gene of interest associated with the nucleocapsid; and a membrane-~eso~ tecl xenotropic protein which determines a host range.
In another plerelled form ofthe present invention, the membrane-associated 25 xenotropic protein ofthe vector particles is a chimeric or hybrid protein inr.l~ltling an exterior receptor binding domain and a membrane-associated domain from a xenotropic envelope protein, at least a portion of the exterior receptor binding domain being derived from a dirrel en~ origin than at least a portion of the Illell,bl ~le-associated domain. The chimeric protein is preferably derived from two origins, wherein no more than one of the two origins 30 is retroviral.
Another embodiment of this aspect of the present invention concerns cell lines that produce the foregoing vector particles. Preferably, such cell lines are stably transfected with a nucleic acid molecule encoding the membrane-associated protein, whose expression is driven by an inducible promoter.

SUBSTITUTE SHF~T (RULE 2~i) Retroviral particles according to the invention may be targeted to a specific cell type by including in the retroviral particles a component, most frequently a polypeptide or carbohydrate, which binds to a cell surface receptor specific for that cell type. Such targeting may be accomplished by plepalil~g a p~ ing cell line which expresses a chimeric env protein comprising a portion of the env protein required for viral particle assembly in conjunction with a cell-specific binding domain. In another elld~dilllcllL, e7rv ~ ,;llS fro more than one viral type may be employed, such that resultant viral particles contain more than one species of env proteins. (See WO 91/02805 entitled "Recombinant Retroviruses Delivering Vector Constructs to Target Cells" and WO 95t3 1566 entitled "Compositions and Methods ffir Targeting Gene Delivery Vehicles", both of which are hereby incorporated by I erel ence.) Yet another embodiment involves inclusion of a cell specific ligand in the retroviral capsid or xenotropic envelope to provide target specificity. In a plerellèd embodiment at this aspect of the invention, the xenotropic env gene employed encodes all or a portion of the xenotropic env protein required for retroviral assembly in conjunction with a receptor binding domain of a polypeptide ligand known to interact with a cell surface receptor whose tissue distribution is limited to the cell type(s) to be targeted, e.g, a hematopoietic stem cell. In this regard, it may be preferable to utilize a receptor binding domain which binds leceplOl~ expressed at high levels on the target cell's surface.
Non-viral membrane-associated proteins may also be used to ~nh~nce targeting of recollll,ill~l~ retroviral particles to hematopoietic stem cells. Representative examples include polypeptides which act as ligands for hematopoietic stem cell surface receptors.
Depending on the tissue distribution of the receptor for the protein in question, the recombinant retroviral particle could be targeted to a dirrèl ell~ subset of hematopoietic stem cells.
When a ligand to be in~ ded within the xenotropic envelope is not a naturally occurring membrane-associated protein, it is necçss~ry to associate the ligand with the membrane, preferably by making a "hybrid" or "chimeric" envelope protein. It is important to understand that such hybrid envelope proteins can contain extr~cP~ r domains from proteins other than other viral or retroviral env proteins. To accomplish this, the gene coding for the ligand can be functionally combined with sequences coding for a membrane-associated domain of the env protein. By "naturally occurring membrane associated protein", it is meant those proteins that in their native state exist in vivo in association with lipid membrane such as that found associated with a cell membrane or on a viral envelope.
As such, hybrid envelopes can be used to tailor the tropism (and effectively increase titers) of 3 5 a retroviral vector according to the invention, as the extrac~llt-l~r component of enl proteins SVBST~TUTE SHFET (RULE 26) WO 96/33281 PCTfUS96/05432 is responsible for specific receptor binding. The cytoplasmic domain of these proteins, on the other hand, play a role in virion formation. The present invention recognizes that numerous hybrid em~ gene products (i.e., specifically, re~LI ~Vil~l env proteins having cytoplasmic regions and extr~c.oll~ r binding regions which do not naturally occur together) S can be generated and may alter host range specificity.
In a p, ~r~ c~: .. ~, this is acco.. ~ ,ulld~ llg the gene codin~ for the ligand (or part thereof c(jllr~ llg receptor binding activity) ~1 ~x;, . .~ e of the membrane-binding domain of the envelope proteins that stably assemble with a given capsid protein.
The resulting construct will code for a bifunctional ~.him~ric protein capable of ~nh~nr.ed cell 10 targeting and inclusion in a retroviral lipid envelope.
Vector particles having non-native mellll,l~e-associated ligands as described herein, will, advantageously, have a host range detel lllilled by the ligand-receptor interaction of the membrane-associated protein. Thus, for targeted delivery to hematopoietic stem cells, a vector particle having altered host range can be produced using the methods of the present 15 invention. The ligand will be sP1ected to provide a host range inrl~lrling hematopoietic stem cells. Many diLrele~ targeting strategies can be employed in co~ ;l;on with this aspect of the invention. For r.7~ ., there are a number of progenitor cell types found in bone III~III~JW that diLrt:lenLiate into blood cells. Many blood cells have relatively short life spans and therefore progenitor cells must cnntin~ ly divide and .li~l ellLiate to replace the lost 20 cells. In a ~l erel I ed embodiment, gene therapy targets helllaLopoietic progenitor cells, inç~ lin~ pluripotent stem cells. These progenitor cells are known to have unique cellular detellllillallL~ that perrnit histological i~lPntific~tion, separation from other cell types by various techniques, inch~t~ing fluolescence activated cell sorting (FACS) and positive and negative selection [see U.S. Patent No. 5,061,620], and which can be used as cell receptors 25 for the membrane-associated proteins of the vector particles of the present invention.
As used herein, a hematopoietic stem cell is a prirnitive, or ;Illlll;~ , cell capable of self-renewal and which is capable of di~er~ g into precursor cells of all hematopoietic lineages, i.e., they are said to be "pluripotent." RecolllbinallL vectors according to the invention may be introduced into such cells or any their more di~re~ ed progeny, such as the various primitive progenitors and the more lineage c~.. l;lled precursor cells that give rise to the various hematopoietic cell lin~g~c. One marker for such early hematopoietic cells is CD34, which can be identified using monoclonal antibodies. See U.S. patent 4,714,680;
WO 93/25216, published December 23, 1993. WO 93/25216 describes a class of hematopoietic stem cells as having the phenotype CD34+/CD38-/HLA-DR- and lacking the 3~ lineage committed antigens CD33, CD10, CD~, and CD71 Representative examples of anti-SUBSmUTE SH EET tRULE Z6) WO 96/33281 PCTIUS96/0',432 CD34 antibodies include 12.8 (Andrews, et al, Blood, 67:842, 1986) and MylO (Civin, et al., J. Immunol., 133:157, 1984, commercially available from Becton Dickinson under the deei~n~tion HPCA-2). Other antibodies may be also utilized to target a selected cell type, such as anti-CD4 antibodies to target CD4+ T-cells and anti-CD8 antibodies to target CD8+
cells (see generally, Wilchek, et al., Anal. Biochem., 171:1, 1988).
The vectors may be constructed to target these cell types for gene delively by in~ tlin~ an ~ es~ le gene which encodes a lllt;mbl~ile-associated protein that binds to a unique cellular determinant of such hematopoietic progenitor cell types. Fx"mrles of such progenitor cell types which could be targeted using recollll~ allL retroviral particles of the 10 present invention include pluripotent stem cells, erythroblasts, lymphoblasts, myeloblasts and meg~k~ryocytes.
Those in the art will also recognize that it is also possible to add ligand molecules exogenously to the leLlovil~l particles which are either incorporated into the lipid envelope or which can be linked chemically to the lipid or protein con.etit~l~nte thereof.
Targeting a retroviral vector carrying a gene of interest to a predt::lellllilled locus on a chromosome may also be employed. Clear advantages of such ~ ,elillg include avoidance of insertional mllt~.neeie and assuring integration at sites known to be transcriptionally active. Techniques for targeting proviral integration to specific sites include inLe~ se modification. See U.S.S N. 08/156,789, supra.
It is further envisioned that the therapy of the present invention be ~el rOl Illed in vitro. For in vitro therapy (also referred to as "ex vivo therapy"), cells are removed and tr~ned~lce~l in vitro. For recolllbill~l~ particles having membrane-associated proteins to enhance hematopoietic stem cell targeting, the need to purify the cells to be targeted in vitro is optional because the vector would specifically tran~d~ce only the targeted cells. Thus, 25 bone marrow samples could be removed from a subject and the desired cell type tr~n~d~ced without the need to perform one or more cell sorting procedures. The tr~n.e~ced cells could then be returned to the same patient or one who is HLA matched.
In addition a wide variety of high affinity binding pairs can be used as targeting elements. Representative examples of include biotin/avidin with an afflnity (KD) of 10-15 M
30 (Richards,Meth Enz., 18~:3, 1990;Green,Adv. inProteinChem., 29:85, 1985)and cystatin/papain with an affinity of 10-14 M (Bjork, etal., Biochemistr~, 29:1770, 1990).
A wide variety of other high affinity binding pairs may also be developed, for example, by preparing and selecting antibodies which recognize a selected hematopoietic stem cell antigen with high affinity (see generally, U.S. Patent Nos. RE 32,011, 4,902,614, " 4.543.439. and 4,411,993; see also Monoclonal Antibodies, Hybridomas: A Neu Dimension SUE~STITUTE SHEET (RULE 26) , in Biological Analyses, Plenum Press, K~nnPtt, McKearn, and Bechtol, eds., 1980, and Antibodies: A Laboratory Manual, Harlow and Lane eds., Cold Spring Harbor Laboratory Press, 1988). The binding pair for such antibodies, typically other antibodies or antibody fr?~mr.ntc, may be produced by reco. l .l.i.~ techniques (see Huse, et al., Science, 246: 1275, 1989; see also Sastry, et al., Proc. Natl. Acad Sci. USA, 86:5728, 1989; and Michelle ~}ting-Mees, et al., Strategies in Molecular Biology, 3: 1, 1990).
As will be evident to one of oldh-aly skill in the art given the disclosure provided herein, either member (or molecule) of the affinity binding pair may be coupled to the retroviral particle. Nevertheless, within p-ert;.,ed embodiments ofthe invention, the larger of the two affinity binding pairs (e.g, avidin of the avidin/biotin pair) is coupled to the retroviral particle. As utilized within the context of targeting, the term "coupled" may refer to either noncovalent or covalent interactions, although generally covalent bonds are pl~;r~;:lled.
Numerous coupling methods may be utili7e-1, inr~ ing, for example, use of crocclinking agents such as N-s~lcr.inimi-1yl-3-(2-pyridyl dithio) propionate ("SPDP"; Carlson, et al., J.
Biochem., 173:723, 1978) and other such compounds known in the art.
In particularly p-~re--~d embodiments ofthe invention, a ~--t;~ er ofthe high afflnity binding pair is either expressed on, or inrl~lded as an integral part of, a retroviral particle, e.g, in the .t;llov,.,ll lipid envelope. For example, a member ofthe high affinity binding pair may be co ~,~.essed with the envelope protein as a hybrid protein or expressed from an 20 app- u~- ;ate vector which targets the ~-.el)~l of the high afflnity binding pair to the cell membrane in the proper orientation.

Uses of Recombinant Retroviral Particles The efficient use of . ~coml~indl.~ retroviral particles to infect cells is dependent on the tropism of the envelope protein expressed on the surface of the retroviral particle . The use of a envelope enables infection of cells from di~. e--~ species.
In one aspect, the present invention provides methods for inhibiting the growth of a selected tumor ("cancer") in a human, comprising the step oftr~nc-il-ring hematopoietic stem cells ex vivo with a vector construct which directs the expression of at least one anti-tumor agent. Within the context of the present invention, "inhibiting the growth of a selected tumor" refers to either (1) the direct inhibition of tumor cell division or met~tctsic7 or (2) immune cell mediated tumor cell Iysis, or both, which leads to a suppression in the net expansion of tumor cells. Inhibition of tumor growth by either of these two meçh~nicmc may 3 ~ be readily deterrnined by one of ordinary skill in the art based upon a number of well known SU~STITUTE SHEET (RULE 26~

-methods, for example, by measuring the tumor size over time, such as by radiologic im~gin~
methods (e.g., single photon and positron emission computerized tomography; see generally, "Nuclear Medicine in Clinical Oncology," Winkler, C. (ed.) Springer-Verlag, New York~
1986) or by a variety of im~ging agents, inr.lll~ing, for example, conventional im~ging agents 5 (e.g, Gallium-67 citrate) or spec~ 7ed reagents for metabolite im~gin~, receptor im~ging, or imm-lnologic im~ ing In addition, non-radioactive meLllods suc~ as ul~a~7uu~ see, "Ultrasonic Di~enellLial Diagnosis of Tumors", Kossoffand Fukuda, (eds.), Igaku-Shoin, New York, 1984), may also be utilized to ~:stim~te tumor size. Alternatively, for other forms of cancer, inhibition of tumor growth may be determined based upon a change in the 10 presence of a tumor marker, e.g, prostate specific antigen ("PSA") for the detectinn of prostate cancer (~e U.S. Patent No. Re. 33,405), and carcino-e~ -yonic antigen ("CEA") for the detection of colorectal and certain breast cancers. For yet other types of cancers such as lPuk~.mi~, inhibition of tumor growth may be determined based upon decreased numbers of lel-k~mic cells in a representative blood cell count.
Within the context of the present invention, "anti-tumor agent" refers to a compound or molecule which inhibits tumor growth. Rep.ese.l~a~ e ~x~mples of anti-tumor agents include immlme activators and tumor proliferation inhibitors. Briefly, immlme activators function by illlplOViilg immlme recognition oftumor-specific ~ntig~n~ such that the immllne system becomes "primed." Priming may consist of Iymphocyte proliferation, di~èn ell~ia~ion~
20 or evolution to higher affinity interactions. The immlme system thus primed will more effectively inhibit or kill tumor cells. Tmmllne activation may be subcategc,-,Ged into immune modulators (molecules which affect the interaction between Iymphocyte and tumor cell) and Iymphokines, that act to proliferate, activate, or di~elell~ia~e immlln~ effector cells.
Representative examples of immllne modulators include CD3, ICAM-l, ICAM-2, LFA-l, 25 LFA-3, ~-2-microglobulin, chaperones, alpha i~e~relon and gamma interferon, B7/BB 1 and major histocompatibility complex (MHC). Representative examples of Iymphokines include gamma interferon, tumor necrosis factor, IL- 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-l 1, GM-CSF, CSF-l, and G-CSF. In addition, RNA molecules having intrinsic biological activity may be utilized as anti-tumor agents.
Sequences which encode anti-tumor agents may be obtained from a variety of sources. For example, plasmids that contain sequences which encode anti-tumor agents may be obtained from a depository such as the American Type Culture Collection (ATCC, Rockville, MD), or from commercial sources such as British Bio-Technology Limited (Cowley, Oxford, Fngl;~n~). Alternatively, known cDNA sequences which encode anti-3 5 tumor agents may be obtained from cells which express or contain the sequences.

SUBSTITUTE S}~EE~ (RULE 263 Additionally, cDNA or mRNA libraries from specific cell sources can be purchased from commercial sources from which the desired sequences can be readily cloned by conventional techniques, e.g., PCR amplification. Sequences which encode anti-tumor agents may also be syntheci7er1, for example, on an Applied Biosystems Inc. DNA synthe.ci7~r (e.g, ABI DNA
5 synth~i7Pr model 392, Foster City, CA).
In addition to the anti-tumor agents des~,lil,ed above, the p~sent illvellLiull also provides anti-tumor agents which comprise a fusion protein of, for example, t,vo or more cytokines, immlme modulators, toxins or diLrtlellLiaLion factors. Plerelled anti-tumor agents in this regard include alpha inLelrelon - Interleukin-2, GM-CSF ~ 1, GM-CSF - IL-2, GM-CSF - IL-3 (see U.S. Patent Nos. 5,082,927 and 5,108,910), GM-CSF - gamma interferon, and gamma illLelrelon - IL-4, with gamma hlLelrelull - interleukin-2 being particularly plerelled.
Within another emborlim~nte the anti-tumor agent may further comprise a membraneanchor. The lllelllbl~le anchor may be sPlected from a variety of sequences, in~.lnriing for 15 example, the Ll;1n~ e domain of well known proteins. Generally, membrane anchor sequ~nces are regions of a protein that anchor the protein to a lll~lllbl~e. Customarily, there are two types of anchor sequPn~s that attach a protein to the outer surface of a cell membrane: (1) Ll;tn~ hl~le regions that span the lipid bilayer ofthe cell membrane (proteins co..~ i..g such regions are referred to integral membrane proteins); and (2) domains which interact with an integral membrane protein or with the polar surface of the membrane (such proteins are referred to as peripheral, or extrinsic, proteins).
Membrane anchors derived from integral membrane proteins are plerell~d.
Membrane sp~nninp: regions typically have a similar structure, with a 20 to 25 amino-acid residue portion consisting almost entirely of hydlul)hobic residues located inside the membrane (see Eisenberg et aL, Ann. Rev. Biochem. 53:595-623, 1984). Membrane ~.~....;..~ regions typically have an alpha helical structure (see Eisenberg et aL at 20; Heijne and Manoil at 109). Within a plerelled embodiment, a membrane anchor is fused to the C-terminus of gamma hlLe~ r~. on fusion protein, wherein the lllelllbl ~le anchor comprises the gamma-chain of the Fc receptor.
Tumorigenicity of an anti-tumor agent can be ~ccec.ced by various assays.
Representative assays include tumor formation in nude mice or rats, colony formation in soft agar. and preparation oftransgenic ~nim~lc such as transgenic mice. In addition to tumorigenicity studies, it is generally preferable to determine the toxicity of an anti-tumor agent. A variety of methods well known to those of skill in the art may be utilized to SU~STITUTE SHEET (~ULE 26) measure such toxicity, inr.lllrling for ~ mple~ clinical r.hrmi.~try assays which measure the systemic levels of various proteins and enzymes, as well as blood cell volume and number.
Once an anti-tumor agent has been srleete~1, it is placed into a vector construct according to the invention.
Such a vector construct can then be packaged into a recombinant retroviral vector and be used to tr~n~ ce ex vivo hematopoietrc stèm c~s w~c~ are then r~ rtroduc~ iTrt~
the patient. In the context of the present invention, it should be understood that the removed cells may not only be returned to the same patient, but may also be utilized to inhibit the growth of selected tumor cells in another allogeneic human.
Pl ~,oa- ~Lion and Purification of Recolll~illalll Retroviral Particles Another aspect of the invention conct;llls the pl t7p~ ~Lion of recolllbillall~ retroviral particles. Recollll)il~alll retroviral particles acco~ g to the invention can be produced in a 15 variety of ways, as those in the art will appreciate. For example, producer cells, i. e., cells co,.l~ ;"g all necessary components for retroviral vector p~rL~ ing (inr~ ing a nucleic acid molecule encoding the lt;LIovil~l vector), can be grown in roller bottles, in bioreactors, in hollow fiber appa~ s, and in cell hotels. Cells can be ,~ illed either on a solid support in liquid medium, or grown as suspensions. A wide variety of bioreactor configurations and 20 sizes can be used in the practice of the present invention.
Cell factories (also termed "cell hotels") typically contain 2, 10, or 40 trays, are molded from virgin polystyrene, treated to provide a Nuclon DTM surface, and assembled by sorlic welding one to another. Generally, these factories have two port tubes which allow access to the chambers for adding reagents or removing culture fluid. A 1 0-layer factory provides 6000 cm2 of surface area for growing cells, roughly the equivalent of 27 T-225 flasks. Cell factories are available from a variety of m~nl-f~ctllrers, inr.~ iing for example Nunc (Baxter, Sanform, ME). Most cell types are capable of producing high titer vector for 3 to 6 days, allowing for multiple harvests. Each cell type is tested to determine the optimal harvest time after seeding and the optimal number of harvest days. Cells are typically initially grown in DMEM supplrm~onted with 2 to 20% FBS in roller bottles until the required number of cells for seeding a cell factory is obtained. Cells are then seeded into the factories - and 2 liters (L) of culture supernatant cont~ining vector is harvested later at an appl Upl iate time. Fresh media is used to replenish the cultures.
Hollow fiber culture methods may also be used. Briefly, high titer retroviral production using hollow fiber cultures is based on increasing viral concentration as the cells SUBSmUTE S}~EET tRULE 26) are being cultured to a high density in a reduced volume of media. Cells are fed nutrients and waste products are diluted using a larger volume of fresh media which circulates through the lumen of numerous capillary fibers. The cells are cultured on the exterior spaces of the capillary fibers in a bioreactor cha.l,l el where cell waste products are ~x~h~nged for S nutrients by diffusion through 30 kD pores in the capillary fibers. Retroviruses which are pr~duced from the cell lines are too large to pass Lhluugh the p~res, arld thus conce-lLIaLe in the hollow fiber bioreactor along side of the cells. The volume of media being cultured on the cell side is appru~;...~t~ly 10 to 100 fold lower then volumes required for equivalent cell densities cultured in tissue culture dishes or flasks. This decrease in volume inversely correlates with the fold induction of titer when hollow fiber retroviral titers are compared to tissue culture dishes or flasks. This 10 to 100 fold induction in titer is seen when an individual leLIovil~l producer cell line is amiable to hollow fiber growth conditions. To achieve m~cimllm cell density, the individual cells must be able to grow in very close proximity and on top of each other. Many cell lines will not grow in this fashion and retroviral pac~ ing cell lines based on these types of cell lines may not achieve 10 fold increases in titer. Cell lines which would grow very well would be non-adherent cell line and it is believed that a l~;LIuvil211 producer line based on a non-adherent cell line may reach 100 fold increases in titer colll~ d to tissue culture dishes and flasks.
Regardless ofthe l~llovil~l partide and production method, high titer (from about 107-10ll cfu/mL) stocks can be pl~ed that will cause high level expression ofthe desired products upon introduction into appropllaLe cells. When all colllponenLs required for retroviral particle assembly are present, high-level cA~ ;s:iion will occur, thereby producing high titer stocks. And while high titer stocks are pl~r~ d, retroviral preparations having titers ranging from about 103 to lo6 cfu/mL may also be employed, although retroviral titers can be increased by various purification methods, as described below.
After production by an al)pro,~,liaLe means, the infectious recombinant retroviral particles may be preserved in a crude or purified form. Crude retroviral particles are produced by cultivated infected cells, wherein retroviral particles are released from the cells into the culture media. The virus may be preserved in crude forrn by first adding a sufficient 30 amount of a formulation buffer to the culture media COI~;"i"g the recolllbhlallL virus to form an aqueous suspenslon.
Recombinant retroviral particles can also be preserved in a purified forrn. Morespecifically, prior to the addition of formulation buffer, the crude retroviral preparation described above is clarified by passing it through a filter. and then concentrated. such as by a cross flow concentrating system (Filtron Technology Corp.~ ~ortborough. MA). Within one SUBSTITUTE SI~EET tRULE 26) embodiment, DNase is added to the concentrate to digest exogenous DNA. The digest is then diafiltrated to remove excess media components and establish the reco.l.billa-l~ virus in a more desirable bu~led solution. The diafiltrate is then passed over a gel filtration column, such as a Sephadex S-500 gel column, and the purified reco..ll,i.,a,lL virus is eluted.
Crude recc""bi"a"~ retroviral ~ ,a,~Lions can also be purified by ion exchange column cl~.c,...alography, such as is described i~ more detail in ~J.S.~.~. Se~zl No. 08/093,436. In general, the crude ~ a-~ion is clarified by passing it through a filter, and the filtrate loaded onto a column col~l;.;.,;..~ a highly sulfonated cellulose matrix, wherein the amount of sulfate per gram of cellulose ranges from about 6 - 15 ~lg. The reco.--billa--L
10 retrovirus is eluted from the column in purified form by using a high salt buffer. The high salt buffer is then ~xch~n~ed for a more desirable buffer by passing the eluate over a molecular toxclll~ion column. The purified plel~a,~ion may then be form~ ted or stored, preferably at -70~C.
Additionally, the ~ a,d~ions co..l~;..i~.g reco",bil~all~ retroviruses according to the invention can be concentrated during purification in order to increase the titer of recombinallL
retrovirus. A wide variety of methods may be utilized for increasing retroviral concentration, inr.lllrlin~ for example, precirit~tion of reco",l~ al~ retroviruses with ammonium sulfate, polyethylene glycol ("PEG") concentration, concentration by centrifugation (either with or without gradients such as PERCOLLTM, or "cushions" such as sucrose, use of concentration filters (e.g, Amicon filtration, Chicago, IL), and 2-phase separations.
Briefly, to accomplish concentration by precipitation of, ~co",bin~ retroviruses with ammonium sulfate, ammonium sulfate is added slowly to an apl)ropli~e concentration, followed by centrifugation and removal of the ammonium sulfate either by dialysis or by separation on a hydrophobic column.
Alternatively, recol"l)inan~ retroviruses may be concentrated from culture m~Aium with PEG (Green, et al., PNAS 67:385, 1970; Syrewicz, et al., Appl. Micro. 24:488, 1972).
Such methods are rapid, simple, and inexpensive. However, like ammonium sulfate precipitation, use of PEG also concentrates other proteins from solution.
Within other embodiments, recombinant retroviruses may be concentrated by centrifugation, and more particularly, low speed centrifugation, which avoids difficulties associated with pelleting that accompanies high speed centrifugation (e.g., virus destruction - or inactivation).
Recombinant retroviruses according to the invention may also be concentrated by an aqueous two-phase separation method. Briefly, polymeric aqueous two-phase systems may be prepared by dissolving two different non-compatible polymers in water. Many pairs of SUBSTlTUTE SHE~ (RULE 26) W O 96/33281 PC~rrUS96/05432 water-soluble polymers may be utilized in the construction of such two-phase systems, in~ ling for ~x~mpl~ PEC~ or methylcellulose, and dextran or dextran sulfate (see Walter and Johansson, Anal. Biochem. 1~5:215, 1986; Albertsson, "Partition of Cell Particles and Macromolecules" Wiley, New York 1960). As described in more detail below in Example 7, lltili7ing PEG at concentrations ranging from 5% to 8% (pler~l~bly 6.5%), and dextran sulfate at conct;llLI~Llolls I~ from 0.4% to 1% ~ler~l~ly 0.4%), an aqueoustwo-phase system may be established suitable for purifying recombinant retroviruses. Utilizing such procedures, appLo~in-~e 100-fold concentration can be achieved with yields of apprc-x;~ y 50% or more ofthe total starting retrovirus.
For purposes of illustration, a representative conc~ lion process which combinesseveral concentration steps is set forth below. Briefly, recolll~ L retroviruses may be pl~aled either from roller bottles, cell f~ctori~c, or bioreactors prior to concentration.
Removed media co--~ the leColllbhl~lL l~lluvilLls may be frozen at -70~C, or more preferably, stored at 2~C to 8~C in large pooled batches prior to processing.
For material obtained from a bioreactor, the recollll)illanl retrovirus pool is first clarified through a 0.8 ~lm filter (1.2 ~lm glass fiber pre-filter, 0.8 ~m cellulose acetate) conn~ctecl in series with a 0.65 ~m filter. This filter all,.,~ nt provides applox;lll~l~ly 2 square feet (sq. ft.)of filter, and allows processing of about 15-20 L of pooled material before clogging. For material obtained from roller bottles or cell factories, a single 0.65 ~lm 20 cartridge (2 sq. ft.) normally suffices for volumes up to 40 L. For 80 L cell factory processes, a S sq. ft. filter may be required.
Preferably, after clarification the filter is rinsed with buffer (e.g, 150 mM NaCI, 25 mM Tris, pH 7.2-7.5). Following clarification"~colllbillall~ retroviruses are concentrated by tangential flow ultrafiltration utili7ing c~c.cettes with a 300,000 mw cut o~ For bioreactor material (co"~;.. ;.. ~ 12% to 16% ~iBS), 4 to 5 L of material may be conce~LI~Led per cassette. For roller bottles or cell factories at 12 to 16% FBS, S to 6 L of material may be concentrated per c~csette. Finally, for cell factories co.,l~;";,.~ 10% FBS, 8 to 9 L of material may be concentrated per cassette. Utilizing such procedures at an app- up~iate pressure dirrel e"lial between filtrate and retçnt~tç, up to 80 L of material may be 30 concentrated to a volume of less than 500 mL in under two hours This process also provides a yield of about 80%.
Following the ultrafiltration step, DNAse may be added to a concentration of 50 U/mL, and recirculated at a lower pump speed with the filtrate line closed for 3~ minutes.
Discontinuous diafiltration is then accomplished by adding additional buffer and utili7inp the SUBSmUTE ~EE~ ~RULE 26) same cross diLrel ellLial pressure as before. Generally, recovery after this step is al.~.loxilllately 70%.
Concentrated material is then subjected to column clll.nla~ography on a Pharmacia S-500 HG size ~r.~ iQn gel"~tili7.in,~ 50 mM NaC1 and 25 mM Tris pH 7.2-7.5 as minimllm 5 salt and ionic ~Llt;ll~LII concentrations. Generally, recolllbill~lL retroviruses elute offin the ~irst peak.
Tangential flow filtration may once again be utilized to further reduce the volume of the prel~alaLion, after which the concell~ ed material is st~?rili7~d by filtration through a 0.2 ~lm Millipore filter (Philadelphia, PA).
As an alternative to in vivo production, the retroviral pa~ gin~ proteins may beproduced, together or separately, from app. opliate cells. However, instead of introducing a nucleic acid molecule enabling production of the viral vector, an in vi~ro pac~ in~ reaction is con~ cted comprising the gag, pol, and xeno env proteins, the retroviral vector, tRNA, and other n~c~e.~ry components. The resulting retroviral particles can then purified and, if 15 desired, concen~l a~ed.

Formulation Of Pharm~.e~ltical Compositions Another aspect of the invention relates to pharm~ce~ltic~l compositions comprising 20 recolllbilldllL retroviral vectors as described above, in combination with a pharm~cel-tically acceptable carrier or diluent, while another aspect is directed toward a method for preserving an infectious recol"~ina~lL retroviruses for subsequent reconstitution such that the recombinant retrovirus is capable of infectin~ m~mm~ n cells upon reconstitution. The methods described can be used to preserve a variety of ~li~e~ viruses, including25 recombinant type C retroviruses such as gibbon ape leukemia virus, feline le~lk~mi~ virus and xeno-, poly- and amphotropic murine le-lk~mi~ virus (Weiss, etaL, RNA Tumor Viruses, 2d ed. 1985). See U. S . S .N. 08/153 ,342.
Pharrn~re~-tically acceptable carriers or diluents are nontoxic to recipients at the dosages and concentrations employed. Representative examples of carriers or diluents for 30 injectable solutions include water, isotonic saline solutions, preferably buffered at a physiological pH (such as phosphate-buffered saline or Tris-buffered saline), mannitol, dextrose, glycerol, and ethanol, as well as polypeptides or proteins such as human serum albumin (HSA). A particularly p, ~r~., ed composition comprises a recombinant retrovirus in 10 mg/mL mannitol, 1 mg/mL HSA, 20 mM Tris, pH 7.2, and 150 mM NaCI. In this case 3 5 since the recombinant retroviral particle represents approximately 1 llg of material it mav be SUE~STI~UTE S~E~ tRULE 2~i) less than 1% of high molecular weight material, and less than 1/100,000 ofthe total material (inr.llltlin~ water). This composition is stable at -70~C for at least six month.c Pharm~ce~1tical compositions ofthe present invention may also additionally include factors which stim-ll~te hematopoietic stem cell division, and hence, uptake andincorporation of vector constructs according to the invention.
Particularly ple;relled methods and compositions for preserving IccollllJillallLretroviruses are described in U.S.S.N. 08/135,938, filed October 12, 1993, and U.S. Serial No. 8/153,342, filed November 15, 1993.
The use of recol"~i.,dl,L retroviruses to tr~ncd~lce he,l.a~opoietic stem cells useful in ~ 10 treating patients requires that the product be able to be transported and stored for long periods at a desired temperature such that infectivity and viability of the recol"bi~
lc;LIov~ S iS retained. The difficulty of preserving recol"bil~ retroviruses absent low te",p~ re storage and transport presents problems in Third World countries, where adequate refrigeration capabilities are often lacking.
The initial stabilization of materials in dry form to the preservation of antitoxins, antigens and bacteria has been described (Flosodort, et al., J. Immz~nol., 29:389, 1935).
However, a limit~tinn in this process inr.l~lded partial denaturation of proteins when dried from an aqueous state at ambient tempel~ul~s. Drying from the frozen state helped reduce this denaturation and led to efficient preservation of other biological materials, inr.llltlin~
bacteria and viruses (Stamp, et al., J. Gen. Microbiol., 1:251, 1947; Rowe, et al., Virology, 42:136, 1970; and Rowe, etal., Cryobiology, 8:153, 1971). More recently, sugars such as sucrose, raffinose, glucose and trehalose were added in various col"l,i"d~ions as stabilizing agents prior to Iyophili7~tion of viruses. The use of sugars enh~nced recovery of viable viruses, for research purposes which require that only some virus survive for later propagation.
Recombinant retroviruses acco, ding to the invention can be stored in liquid, orpreferably, Iyophilized form. Factors inflllen(~inE stability include the formulation (liquid, freeze dried, conctit~nt.c thereof, efc ) and storage conditions, inr.~ inp temperature, storage container, exposure to light, etc Alternatively, retroviral particles according to the invention can be stored as liquids at low temperatures. In a pl~r~ d embodiment, the recon.l)i,.al,L retroviruses ofthe invention are formlll~ted to preserve infectivity in a Iyophilized form at elevated temperatures, and for this form to be suitable for injection into patients following reconstitution.
Recombinant retroviral particles comprising retroviral vector constructs according to 3~ the invention can be formulated in crude or, preferably, purified form. Crude retroviral SUBS I l l ~JTE SHF~T tRUL~ 26) pl ~pa- dLions may be produced by various cell culture methods, where retroviral particles are released from the cells into the culture media. RecolllbillanL retroviral particles may be preserved in crude form by adding a sufficient amount of formulation buffer. Typically, the formulation buffer is an aqueous solution co,.~ ;"g various components, such as one or 5 more saccharides, high molecular weight structural additives, buffering components, and/or amino acids.
The recombillallL retroviruses described herein can also be preserved in a purified form. For in.et~ncP, prior to the addition of formulation buffer, crude plepaldlions as described above may be clarified by filtration, and then concentrated, such as by a cross flow 10 concentrating system (Filtron Technology Corp., Nortborough, MA). DNase may be added to the concentrate to digest exogenous DNA, followed by diafiltration to remove excess media colllponellLs and substitute in a more desirable buffered solution. The diafiltrate may then passed over a gel filtration column, such as a SephadexTb' S-500 gel column, and the eluted retroviral particles retained. A suffficient amount of form~ tion buffer may then be 15 added to the eluate to reach a desired final concentration of the con.ctit~lPnts and to minim~lly dilute the retroviral pl ClJdl d~ion. The aqueous suspension can then be stored, preferably at -70~C, or immPrli~t~Ply formlll~te-l In an alternative procedure, the crude ~le~Jdld~ion can be purified by ion P.x~h~nge column clllullldLography, as described in co-owned U.S.S.N. 08/093,436, filed July 16, 1993.
20 Briefly, the crude recolllbinallL retrovirus is ~ rified by filtration and then loaded onto a column comprising a highly sulfonated cellulose matrix. Highly purified l~coll~billallL
retrovirus is eluted from the column using a high salt buffer, which is then exchanged for a more desirable buffer by passing the eluate over a molecular exclusion column. After recovery, formulation buffer may then added to adjust the final concentration, as discussed 25 above, followed by low temperature storage, preferably at -70~C, or immPfli~tP. formulation.
When a dried formulation is desired, an aqueous pl~pdldLion cont~inin~? a crude or purified retroviral prepdld~ion can be prepared by Iyophilization or evaporation.
Lyophilization involves cooling the aqueous preparation below the glass transition temperature or below the eutectic point temperature of the solution, and removing water by 30 sublimation. For example, a mlllti~tep freeze drying procedure as described by Phillips et al.
(Cryobiology, 18:414, 1981) can be used to Iyophilize the formlll~ted recombinant virus, preferably from a temperature of-40~C to -45~C. The resulting composition should contain less than 10% water by weight. Once Iyophilized, such a preparation is stable and may be stored at -20~C to 25~C

SL18ST~TUTE S~EEl~ (RULE 26) In an evaporative method, water is removed by evaporation from the retroviral plepal~lion aqueous suspension at ambient temperature. Ev~pol~lion can be accomplished by various techniques, in~ riing spray drying (see EP 520,748), where the pl~pa~Lion is delivered into a flow of preheated gas, usually air, whereupon water rapidly evaporates from 5 droplets of the suspension. Spray drying appal~L.ls are available from a number of m~mlf~ctllrers (e.g, Drytec, ~td., Tonbridge, r.~gl,...d, ~ab-Plant, ~td., Hudders~eid, F.ng]~n~). Once dehydrated, the rec~lllbill~ll retroviral ~ p~-lion is stable and may be stored at -20~C to 25~C. The resnlting moisture content of the dried or Iyophilized ple~ lion may be determined through use of a Karl-Fischer appal~Llus (EM Science10 ~q~l~ct~r' VlB volumetric titrator, Cherry Hill, NJ), or through a gravimetric method. Once dehydrated, the rec~ retrovirus is stable and may be stored at -20~C to 25~C.
As m~ntion~d previously, aqueous prep~l~lions comprising l~lloviluses according to the invention used for formulation are typically composed of one or more saccharides, high molecular weight structural additives, buffering components, and water, and may also 15 include one or more amino acids. It has been found that the colll~illalion of these components acts to preserve the activity of the recolllbill~lL retrovirus upon freezing and Iyophilization, or drying through evaporation. See co-owned U.S.S.N. 08/153,342, filed November 15, 1993. Various saccharides may be used alone or in combination, in~ ing sucrose, m~nnitol, glucose, tr~h~losP, inositol, fructose, maltose, and galactose, with lactose 20 being particularly plt;rell~d. The concentration ofthe saccharide can range from 0.1% to 30% by weight, preferably from about 1 % to 12% by weight. A particularly pl ~;re~l l ed concentration of lactose is 3% to 4% by weight. Additionally, saccharide colllbill~-lions can also be employed, in~lllrling lactose and m~nnitol or sucrose and ~ ;lol. It will also be evident to those skilled in the art that it may be preferable to use certain saccharides in the 25 aqueous solution when the lyophilized forrn--l~tion is int~n-1ed for room temperature storage.
Specifically, ~1ic~c~h~ides~ such as lactose or trehalose, are p~r~ d for such formulations.
One or more high molecular weight structural additives may be used to aid in preventing retroviral aggregation during freezing and provides structural support in the Iyophilized or dried state. In the context of the present invention, structural additives are 30 considered to be of "high molecular weight" if they are greater than 5000 daltons. A
pl ere, I ~d high molecular weight structural additive is human serum albumin (HSA), although other substances may also be used, such as hydroxyethyl-cellulose, hydroxymethyl-cellulose, dextran, cellulose, gelatin, povidone, etc. Preferably, the concentration of the high molecular weight structural additive can range from 0.05% to 20%, with 0.1% to 10% by weight being 3 ~ pl c rt;l I ed, and a concentration of 0.1 % by weight HSA being particularly preferred SUBST~TUTE S~tEEl- (RULE 26) WO 96/33281 PCTtUS96tO5432 Amino acids, if present, tend to further preserve retroviral infectivity. In addition, amino acids function to further preserve retroviral infectivity during sublimation of the cooled aqueous suspension and while in the lyophilized state. A plerelled amino acid is arginine, but other arnino acids such as Iysine, o~ ne, serine, glycine, ~ t~min~, 5 asparagine, gl-lt~mic acid or aspartic acid can also be used. Preferably, the amino acid c~ .,t,~ n ranges from 0.1% to 10% byweight. A ~alLieulally ~lefelle~ ~Ll~,illill~, concentration is 0.1% by weight.
A variety of buffering components may be used to ...~;..l~;.. a relatively constant pH, depending on the pH range desired, preferably between 7.0 and 7.8. Suitable buffers include 10 phosphate buffer and citrate buffer. A particularly p~ere"ed formulation pH is 7.4, and a pl erel l ed buffer is ~
It may also be preferable to include in the formulation a neutral salt to adjust the final iso-osmotic salt conce"L~Lion. Suitable neutral salts include sodium chloride, potassium chloride, and m~gne~illm chloride, with sodium chloride being plerelled.
A particularly ~), erell ed method of preserving, ecollll~ allL retroviruses in a Iyophilized state for subsequent recnn.~tit~ltit n comprises: (a) prel)~i,lg an aqueous reco",bin~,L leLluvil~l plè~ Lion comprising, in ~d-lition to the recombinant retrovirus, about (i) 4% by weight of lactose, (ii) 0.1% by weight of HSA, (iii) 0.03% or less by weight of NaCI, (iv) 0.1% by weight of arginine, and a sufficient amount of trom~oth~min~ to provide a pH of app, uxi~ ely 7.4; (b) cooling the p, epal~LLion to a temperature of about ~0~C to -45~C to form a frozen p,~a"-Lion; and (c) removing water from the frozen p,l;pa~ion by sublimation to form a Iyophilized composition having less than 2% water by weight. It is p~ erel I ed that the recombinant retrovirus be replication defective and suitable for ~flminictration into humans cells upon reconstitution.
The Iyophilized or dehydrated retroviruses of the subject invention may be recon.~tit~lted using a variety of substances, but are preferably recon~tit~ted using water. In certain instances, dilute salt solutions which bring the final formulation to isotonicity may also be used. In addition, it may be advantageous to use aqueous solutions cont~ining components known to ~nh~n~e the activity of the recon~tit~ted virus. Such components include cytokines, such as IL-2, polycations, such as plotamh-e sulfate, or other components which t-nh~nce the tr~n.~duction efficiency of the recon~tit~lted virus Lyophilized or dehydrated recombinant virus may be recon~titllfed with any convenient volume of water or the reconctit~lting agents noted above that allow substantial, and preferably total solubilization of the Iyophilized or dehydrated sample.

SUBS'rlTUTE 5~EFi- tRULE 26) ~1minietration of Recombinant Retroviral Particles In another aspect of the present invention, methods are provided for treating human patients af~licted with a variety of r1iee~e~e~ inrl-lrling a genetic disease, cancer, an infectious 5 disease, an auto;...,....l-~ disease, and infl~ toly disease, a cardiovascular disease, and a degenerative disease. Examples of genetic di.ce~etos include but are not ~mited to;
th~l~e.c~mi~ phenyLketonuria, Lesch-Nyan syndrome, SCID, hemophilia A and B, cystic fibrosis, Du~htqnn~'s mllsclll~r dystrophy, inherited emphysema, familial hypercholesterolemia, and ~ ch~r~s disease. Fx~mrles of cancers include but are not 10 limited to; solid tumors, lellkPmi~e and lyrnphom~c Representative examples include melanomas, colorectal carcinomas, lung calci~lolllas (inrlllr1ing large cell, small cell, squamous and adeno-carcinomas), renal cell carrinnm~e, cervical cancer, adult T-cell Iymphoma lellk~mi~ and breast adeno-carr.inom~e Infectious ~iiee~çs include but not limited to; hPp~titi~, tuberculosis, malaria, human immllnnd~firisncy virus, herpes virus, tetanus, 15 dysentery, shigella, FeLV, and FIV. Degenel~live diee~eee include but are not limited to:
~17htoim~r's disease, mllltirle. sclerosis, mllec~ r ,1Y~LIO~}IY~ yo~ophic lateral sclerosis, Tnfl~mm~tory fliee~ees include ~ oid arthritis, spinal m.oningitie, and pancreatitis.
Autoimmune r~i~e~es include diabeLes, uveitis, HIV, and SCID. Cardiovascular dieç~es inclll(l~, chronic rh~llm~tic heart disease, arteriosclerosis, mitral valve and aortic stenosis, 20 myocarditis, pericarditis, Marfan's syndrome, Ehlers-Danlos syndrome, Churg-Strauss syndrome, and scleroderma.
Each of these methods comprise ~ g to a human a recolllbh~all~ retroviral particle pl e~,al ~Lion as described above, such that a therapeutically efficacious amount of gene product encoded by the gene of interest carried on the vector construct is produced.
25 As used herein, a "therapeutically effective amount" of a gene product expressed from a vector construct according to the invention is an amount that achieves a desired therapeutic benefit in a patient to an extent greater than that observed when the patient was not treated with the gene product. For instance, when the gene product is factor VIII, a "therapeutically effective amount" refers to the amount of factor VIII needed to produce therapeutically 30 beneficial clotting and will thus generally be determined by each patient's ~ttçn-1in~ physician.
although serum levels of about 0.2 ng/mL (about 0.1% of "normal" levels) or more will be therapeutically beneficial. When the gene product is an RNA molecule with intrinsic biological activity, such an ~nti~çnce RNA or ribozyme, a "therapeutically effective amount"
is an amount sufficient to achieve a clinically relevant change in the patient's condition 3 5 through reduced expression of the harmful gene product. most often a protein. In a SlJ8STlTUTE SHEE~ (RULE 2~i) -WO 96/33281 PCT/US96/0~432 p,t;~ed embodiment, the RNA molecule with intrinsic biological activity will be expressed in tran.c(lllced hematopoietic stem cells in molar excess to the targeted RNA molecule.
Expression levels of the heterologous and targeted RNAs can be determined by various assays, e.g., by PCR analysis.
Typically, the dosage for ex vivo gene modified hematopoietic stem cells will be in the range of 105 to 108 cells per kilogram }~atemt body we~ght dc~,r,d,l,g upa~r ~}re purity of stem cells in the starting population. Thus, for example, for CD34+ selected cells, usually 107to 108 cells will be tr~n~d-lced ex vivo for re-infusion into a patient; for more highly enriched stem cell populations, such as CD34+ Thy-l+Lin~ selected cells, usually from 0 105 to 107 cells will be tr~n~duced and re-infused. The stem cell population will usually be tr~n.~d~lced at mutiplicity of infection (MOI) of 10 to 1000, typically around 100 infectious ~eco.~ retroviral particles per cell.
Preferably, high titer p, ~al d~ions of retroviral vectors are used for ex vivo tr~n~ lction. The volume that the high titer ple~dldLion is delivered in ex vivo is preferably 15 not greater than 10% of the culture me~ m volume of the cell culture. More preferably, the volume of the high titer pl c;pa~ dlion is less than 1 %, still more preferably less than 0.1%, and still more pl~ldbly less than 0.001% ofthe total cell culture volume. Additionally the retrovirus is delivered in a m~ m that is free of agents that disturb or are toxic to the tr~n~ ce~ cells in culture (eg. in an aqueous liquid with a composition similar to that of cell 20 culture met1illm).

Hematopoietic Stem Cells A pluripotent hematopoietic stem cell may be defined as follows: (1) a cell which 25 gives rise to progeny in all defined hematolymphoid lineages; and (2) a cell which is capable of fully reconstituting a seriously immunoco"")l ull~ised host of all blood cell types and their progenitors, in~.ln~in~ the pluripotent hematopoietic stem cell by self renewal."Hematopoietic stem cells" refers to a population of hematopoietic cells having all of the long-term engrafting potential in vivo. Animal models for long-term engrafting potential 30 of candidate human hematopoietic stem cell populations include the SCID-hu bone model (Kyoizumi et al. (1992) Blood 79:1704; Murray et al. (1995) Blood 85( ') 368-378) and the in utero sheep model (Zanjani et al. (1992) J. Clin. Invest. 89: 1179. For a review of animal models of human hematopoiesis, see Srour et al. (1992) J. Hematother. 1: 143 - 153 and the references cited therein. At present, the best in vitro assay for stem cells is the long-term 35 culture-initiating cell (LTCIC) assay, based on a limiting dilution analysis ofthe number of SUBSTITUTE SHEE~ ~RULE 263 clonogenic cells produced in a stromal co-culture after 5-8 weeks. Sutherland et al (1990) Proc. Natil Acad. Sci. 87:3584-3588. The LTCIC assay has been shown to correlate with another commonly used stem cell assay, the cobblestone area forming cell (CAFC) assay, and with long-term engrafting potential in vivo. Breems etal. (1994) Lellkemi~ 8:1095.
S For use in the present invention, a highly enriched stem cells population is plt;re lled i~ order to ..~,.x;...;,~ efficiency of gene transferint~ the desired t~rget cells. As des.,,;l,ed more fully below, an enriched stem cell population is PYemrlifiçcl by a population of cells selected by t~A~l eS~iOn of the CD34 marker. In LTCIC assays, a population enriched in CD34+ cells will have an LTCIC frequency in the range of 1/50 to 1/500, more usually in the range of 1/50 to 1/200. Preferably, the stem cell population will be more highly enriched for stem cells than that provided by a population s.olected on the basis of CD34+ t A~Jl es~ion alone. By use of various techniques described more fully below, a highly enriched stem cell population may be obtained. A highly enriched stem cell population will typically have an LTCIC frequency in the range of 1/5 to 1/100, more usually in the range of 1/10 to 1/50.
Preferably it will have an LTCIC frequency of at least 1/50. FYto.mrl~ry of a highly enriched stem cell population is a population having the CD34+Thyl+Lin- phello~y~e as described in U.S. Patent No. 5,061,620. A population ofthis phenotype will t-ypically have an average LTCIC frequency of app.vx""~l~ly 1/20. Murray et al. (1995); Lansdorp et al. (1993) J.
Exp. Med. 177: 1331.
~em~tQpoietic stem cells may be isolated from any known source of stem cells, in~ fling bone marrow, mobilized peripheral blood (MPB), and umbilical cord blood.
Initially, bone n,~"c)w cells may be obtained from a source of bone marrow, inel~ltlinEg ileum (e.g, from the hip bone via the iliac crest), tibia, femur, spine, or other bone cavities Other sources of ht;l.laLopoietic stem cells include e"ll,lyo,fic yolk sac, fetal liver, and fetal spleen.
For isolation of bone lll~lluw, an a~pl~,p,i~Le solution may be used to flush the bone, in~ linpr saline solution, conveniently supplem~nted with fetal calf serum (FCS) or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from about 5 to 25 mM. Convenient buffers include HEPES, phosphate buffers and lactate buffers. Otherwise bone lllallOW may be aspirated from the bone in accordance with conventional techniques.
Methods for mobilizing hematopoietic stem cells into the peripheral blood are known in the art and generally involve tre~tm~nt with chemotherapeutic drugs (e.g., cyclophosphamide or etoposide), cytokines (e.g, GM-CSF, G-CSF or IL-3), or combinations thereof. Typically, apheresis for total white cells begins when the total white cell count reaches 500-2000 cells/ml and the platelet count reaches 50,000/ml In order to SUBSTrr~ iHEET (RULE 26) m~ximi7e hematopoietic stem cell recovery from MPB, daily aphoresis samples may be monitored for cells ~p~ g CD34 and/or Thy- l . Desirably, the collection of peripheral blood leukocytes begins when an increase in CD34+ and/or Thy-l+ cells is detected in order to obtain a sample during the peak of stem cell mobilization. While CD34 t~A~JI ession S normally correlates with stem cell mobilization, in some cases it does not. Therefore, it is prRerable to use the Thy- 1 marker alone or in ~ ;u~ th CD~4 to IllCslli~ul s~em cell mobilization (Murray, ef al. 1995).
Various techniques may be employed to separate the cells by initially removing cells of dedicated lineage ("lineage-committed" cells). Monoclonal antibodies and monoclonal 10 antibody fragments are particularly useful for identifying markers associated with particular cell lineages and/or stages of di~reI~Lialion. The antibodies (or antibody fragments) may be t~hed to a solid support to allow for crude separation. The separation techniques employed should ~ x;~ e the viability ofthe fraction to be collected.
The use of separation techniques include those based on differences in physical 15 (density gradient c~ntrifi~ tinn and counter-flow centrifugal elutriation), cell surface (lectin and antibody afflnity), and vital st~ining properties (mitochondria-binding dye rhodamine 123 and DNA-binding dye Hoechst 33342). Procedures for separation may include m~gnetic separation, using antibody-coated m~gnP,tic beads, afflnity cl,l~,mdLography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, 20 in~ ling complement and ~;y~u~OAinS~ and "panning" with antibody ~tt~ched to a solid matrix or any other convenient technique. Techniques providing accurate separation include flow cytometry which can have varying degrees of sophistication, e.g., a plurality of color ~h~nnçl~ low angle and obtuse light scattering detectin~ çh~nn~.lc impedance channels, etc.
A large proportion of the diLr~ Liated cells may be removed by initially using a25 relatively crude separation, where major cell population lineages of the hematopoietic system such as Iymphocytic and myelomonocytic, are removed, as well as minor populations, such as mt~g~k~ryocytic, mast cells, eosinophils and basophils. Usually, at least about 70 to 90 percent of the hematopoietic cells will be removed.
"Positive selection" refers to a selection procedure whereby the cell population of 30 interest expresses the marker used as the basis for selection and, therefore, cells expressing the marker are retained and those not t~AI~l es~i"g the marker are discarded. "Negative selection" refers to a selection procedure whereby the cell population of interest does not express the marker or have the characteristic that is used as the basis for selection and, therefore~ cells having the specified marker or characteristic are discarded and those not , ~ having the specified marker or characteristic are retained. Thus, positive selection SU8STITUTE SHE~ (RULE 2~i) techniques may be employed to select stem cells based on, e.g, CD34 expression. One technique providing positive selection for CD34+ cells with high purity is described in PCT
patent application No. WO94/02016, in which CD34+ cells are selected using a hapten conjugated anti-CD34 antibody and a hapten competition release system.
Conco~ y or subsequent to a gross separation providing for positive s~olectiQn e.g~, using the CD34 marker, a negative selection ma~r be carried out, w~Tere al~t;b-~-lies to lineage-specific ...~ke.~ present on de~lic~ted cells are employed and the lineage-cr)~ led removed, e.g, by m~gn~tic bead depletion or flow cytometer. For the most part, these markers include CD2; CD3-, CD7-, CD8-, CD10-, CD14-, CD15; CD16-, CD19-, CD20;
10 CD33- and glycophorin A-. Normally, negative selection will yield a cell population which is at least CD14- and CD15-, and preferably which is at least CD2-, CD14-, CD15-, CD16;
CD19- and glycophorin A-. As used herein, Lin~ refers to a cell population lacking at least one lineage-specific marker. The hematopoietic cell composition may then be further separated using positive selection for the Thy-1 marker and/or selection for rhodaminel~, 15 whereby a highly enriched hematopoietic stem cell population is achieved. See Table 1.
The purified helllatopoietic stem cells have low side scatter and low to merliIlm fc.. w~. .1 scatter profiles by FACS analysis. Cytospin p. è~ ions show the enriched stem cells to have a size between mature Iymphoid cells and mature granulocytes. Cells may be selected based on light-scatter properties as well as their ~,A~JI ession of various cell surface 20 antigens.
While it is believed that the particular order of separation is not critical to this invention, the order indicated is plerelled. Preferably, cells are initially separated by a crude separation, followed by a fine separation, with positive selection of one or more markers associated with stem cells and negative selection for one or more markers associated with 25 lineage co~ ed cells. Compositions highly enriched in hematopoietic stem cells may be achieved in this manner. The desired hematopoietic stem cells are exemplified by a population with the CD34+Thy-1+Lin~ phenotype.
A hematopoietic stem cell composition is characterized by being able to be Ied in culture for extended periods of time, being capable of selection and transfer to 30 secondary and higher order cultures, and being capable of difIel e"l ~ting into the various Iymphocytic and myelomonocytic lineages, particularly B and T Iymphocytes, monocytes, macrophages, neutrophils, erythrocytes and the like The stem cells may be grown in culture in an applc,p.iate nutrient medium~ including conditioned medium, a co-culture with an appl op~ iate stromal cell line or a medium 3 5 comprising a combination of growth factors sufficient to m~int~in the growth of SUBSTI~UTE SHE~T (RULE 26) hematopoietic cells. For conc1itit)ned media or co-cultures, various stromal cell lines may be used. Since human stromal cell lines are not required, other stromal cell lines may be employed, inelu(1ing rodentiae, particularly murine stromal cell lines. Suitable murine stromal cell lines include AC3 and AC6, which are described in Whitlock et al. Cell 48:1009, 1987.
Preferably, the stromal cell line used is a passage of AC6, AC6.2 1 (otherwise referred to as S~l).
Cytokines may also be added, in~ inp, e.g, le -k~mi~ inhibitory factor (LIF), interleukins, colony stim~ tin~ factors, and stem cell factor (SCF, also known as steel factor, c-kit ligand, MGF). Of particular interest are LIF, stem cell factor, IL-3, IL-6, GM-10 CSF, G-CSF, MIP-la, the flk2/flt3 ligand, and TPO/npl ligand. The factors which are employed may be naturally occurring or synthetic, e.g, ple~aled recombinantly, and preferably are human. The amount of the factors used will generally be in the range of about 1 ng/ml to 100 ng/ml. Generally, for LIF, the concentration will be in the range of about 1 ng/ml to lO0 ng/mg, more usually 5 ng/ml to 30 ng/ml; for IL-3, the concen~Lion will be in 15 the range of about 5 ng/ml to 100 ng/ml, more usually 5 ng/ml to 50 ng/ml; for IL-6, the concentration will be in the range of about 5 ng/ml to 50 ng/ml, more usually 5 ng/ml to 20 ng/ml, and for GM-CSF, the concentration will generally be 5 ng/ml to 50 ng/ml, more usually 5 ng/ml to 20 ng/ml, and for SCF the concentration will generally be 10 ng/ml to 150 ng/ml, and usually 50 ng/ml to 100 ng/ml.
In one embodiment, the hematopoietic stem cells are optionally exran-led prior to or after retroviral tr~n.~ ction. During expansion, the growth factors may be present only during the initial course of the stem cell growth and ~xp~n~ n, usually at least 24 hours, more usually at least about 48 hours to 4 days, or may be ~ ;.;.led during the course ofthe expansion.
For use in clinical settings, it is preferable to tr~ncdllce the hematopoietic stem cells without prior or subsequent expansion. In one embodiment therefore, the stem cells are cultured with or without cytokines in an apl)lopliate metlillm tr~n~ ced with the appropriate vector, cultured for approximately 72 hours and reintroduced into the host. In one embodiment ofthe invention, at least about 0.1% ofthe hematopoietic stem cells in a 30 given hematopoietic cell population are tr~n.cduced with a recombinant retroviral particle according to the invention. In another embodiment, at least about 1% of the hematopoietic stem cells in a given hematopoietic cell population are tr~nc~l~lced with a recombinant retroviral particle according to the invention, while in yet another embodiment of the invention, at least about 5% of the hematopoietic stem cells in a given hematopoietic cell 35 population are tr~n.~ ced with a recombinant retroviral particle according to the invention.

SUBSTITUTE SHLE~ (RULE 26) In a plcrelled embodiment ofthe invention, at least about 10% ofthe hematopoietic stem cells in a given hematopoietic cell population are tr~n.ed~lced with a recoll,l,i,lalll retroviral particle as described herein. In another ~ler~lled embodiment, at least about 25% ofthe hematopoietic stem cells in a given ht:mdLo~oietic cell population are transduced. In S particularly plercllt;d embo~im~nts, at least about 50% ofthe hematopoietic stem cells in a gr~en h~..,a~opoietic cell popula~ron are L~ ced Ev~ mc~re ~ RII ~d aT~ embodiments where at least about 60%, 70%, 75%, 80%, 90%, or 95% ofthe hematopoietic stem cells in a given hematopoietic cell population are tr~ncd~lced with a lecc,llll~lalll Ic~lovil~l particle as described herein.
Tr~ncd~1ction may be accomplich~d by the direct co-culture of stem cells with recolllbindllL retroviral particle producer cells, e.g., by the method of Bregni, et al., Blood 80:1418, 1992. For clinical applications, however, tr~n.cduction by culturing the hematopoietic stem cells with l~colll~illall~ retroviral S~ ;llld~ alone or with purified retroviral pl cl~ala~ions as described herein, in the absence of stromal cells, is pl cr~ ,d.
15 Tr~nc(luctions may be pelr.~ ed by c~ lring the hclll~lopoietic stem cells with the virus for from about four hours to six days. Preferably, tr~ncductiQn is carried out for three days, with the media replaced daily with media cor.~ g fresh reco~ retrovirus particles.
Alternatively, the stem cells may be cultured in the presence of the retrovirus for several hours, e.g, four hours, daily for three to four days, with fresh media repl~in~ the virus-20 co"l~il-;..~ media each day. In addition, the cell/virus ~l~dlions may be centrifuged.
Typically, growth factors will be inr.l~l-led in such amounts and concellLIaLion to m~int~in cell viability and induce cell cycling. Normally the cultures will include at least stem cell factor (SCF, also known as steel factor, MGF, and c-kit ligand), IL-3, and IL-6. Other cytokines of interest include lellk~mi~ illh~lJiL~ly factor (LIF), G-CSF, GM-CSF, MIP-l, flk2Mt3 ligand, 25 and TPO. Polycations, such as plola"lil,e sulfate, polybrene and the like, will generally be included to promote binding. PI~Jlal,line sulfate and polybrene are typically used preferably at a concentration of 4 ng/ml).
Gene ~,~n~re, into hematopoietic stem cells may be used to treat a variety of neoplastic, infectious or genetic diseases. For example, one may introduce genes that confer 30 rPcict~nre to chemotherapeutic agents, thereby protecting the progeny hematopoietic cells, allowing higher doses of chemotherapy and thereby improving the therapeutic benefit. For instance, the mdrl gene (see U.S Patent No. 5,206,352) may be introduced into hematopoietic stem cells to provide increased resistance to a wide variety of chemotherapeutic drugs which are exported by the mdrl gene product, in combination with 3~ the a~iminictration of chemotherapeutics such as taxol, e.g, for breast cancer treatment.

SUBSTrrUTF. ~HEET tRULE 2~i3 Similarly, genes that provide increased resict~nr,e to alkylating agents, such as melphalan, may be introduced into hematopoietic stem cells in conjunction with high dose chemotherapy.
For viral infections that primarily affect hematolymphoid cells, stem cells may be 5 modified to endow the progeny with recict~nr.e to the infectious agent. In the case of human trnmllnod~i";ell~,y virus ~HIV), for example, s~c, seffse, ~ ..r.~ or~ y~re s~quences may be introduced that illle~ rere with viral infection or replication in the target cells.
Alternatively, the introduced gene products may serve as "decoys" by binding essçnti~l viral proteins, thereby i"~e,f~ g with the normal viral life cycle and inhibiting replication.
Alternatively, hematopoietic stem cells may be modified to produce a product to correct a genetic d~fi~iency, or where the host has acquired a genetic deficiency through a subsequent disease. Genes that may correct a genetic deficiency include adenosine de,min~ce for the tre~tm~nt of ADA- severe combined immllnodeficiency;
glucocerebrosidase for the trç~tm~nt of (~ rh~r's disease; beta-globin for the tre~tm~nt of 15 sickle cell anemia; and factor VIII or factor IX for the tre~tm~nt of hemophilia.
In many situations, cell immllnoLhel~lJy involves removal of bone marrow or other source of hematopoietic stem cells from a human host, isolating the stem cells from the source and optionally ~Ypan~ling the stem cells. Meanwhile, the host may be treated to partially, substantially or completely ablate native hellla~opoietic capability. The isolated 20 hematopoietic stem cells may be modified prior to or during this period of time, so as to provide hematopoietic stem cells having the desired genetic modification. After completion of the tr~tm~nt of the host, the modified hematopoietic stem cells are reintroduced to the host to provide for expression of the foreign gene(s), and to reconstitute a functional hematopoietic system, if necessary. The methods of stem cell removal, host ablation and 5 hematopoietic stem cell repopulation are known in the art. If necessary, the process may be repeated to ensure substantial repopulation of the modified stem cells.
To ensure that the hematopoietic stem cells have been s~ccçccfi-lly modified, a vector-specific probe, or PCR using vector-specific primers, may be used to verify the presence of the vector construct in the tr~n.cduced stem cells or their progeny. In addition, 30 the cells may be grown under various conditions to ensure that they are capable of maturation to all of the hematopoietic lineages while m~int"ining the capability, as appropriate, of the introduced DNA. Various tests in vitro and in vivo may be employed to ensure that the pluripotent capability ofthe stem cells has been m~int~ined.
The compositions comprising hematopoietic stem cells provide for production of 3 5 myeloid cells and Iymphoid cells in appropriate cultures. In each of the cultures. mouse or SUB5mUTE SHEE~ (RULE 2~

human stromal cells are provided, which may come from various sources, incl~ ing but not limited to, AC3, AC6 or stromal cells derived from mouse or human bone Illa,low by selection for the ability to ~ hematopoietic stem cells, and the like. Preferably, the stromal cells are AC6.21 and the ability to produce B Iymphocytes and myeloid cells is 5 determined in cultures supplied with LIF and IL-6. Generally, after 3 to 6 weeks of culture aE~AC6.2 1 stromal cells, the cells are analyzed by F~CS for eAI.re~sion of CDl 9 (a B cell marker) andCD33 (a myeloid cell marker). ~lrlitinn~lly, the he~ opoietic stem cells can be analyzed for the ability to give rise to B cells, T cells and myelomonocytic cells in in vivo assays, as described below.
To demonstrate dirr~l ~;nLiation to T cells, fetal thymus is isolated and cultured from 4 to 7 days at 25~C, so as to deplete substantially the Iymphoid population. The cells to be tested for T-cell activity are then microinjected into the thymus tissue, where the ~A of the population which is injected is mi.em~tcch-od with the ~A ofthe thymus cells. The thymus tissue may then be tr~n.~pl~nted into a scid/scid mouse as described in US Patent No.
5,147,784, particularly tr~n~pl~ntinp: under the kidney c~ps -le. Mice are s~crifi~ed 6 to 7 weeks after tr~n.~pl~nt~tion and the thymus graft recovered and reduced to a single cell suspension. Donor-derived cells are detected by E~A staining and thymus di~lel~ ion analyzed by CD4 and CD8 st~inin~ and FACS analysis.
Further demonstration ofthe s--ct~in~d regenerative ability of hematopoietic stem cell 20 populations may be accompli~hed by the d~tection of continued myeloid and B-lymphoid cell production in the SCID-hu bone model. See Kyoizumi, et al., Blood 79:1704, 1992.Briefly, human fetal bone is i~ol~ted and a Innf~itu~lin~lly sliced portion of this bone is transferred into the ...~ .y fat pad of a SCID/SCID animal. The bone cavity is rlimini~hed in endogenous cells by whole body irradiation of the mouse host prior to injection of the test 25 donor hematopoietic cell population. The E~A of the population which is injected is micm~tçhed with the ~,A ofthe recipient bone cells. Stem cells from human hematopoietic sources will sustain B Iymphopoiesis and myelopoiesis in such a SCID-hu bone model.
To demon:jL,~ the ability ofthe hematopoietic stem cell population to give rise to red blood cells, one may use conventional techniques to identify BFU-E units, for example, 30 methylcellulose culture to show that the cells are capable of developing the erythroid lineage.
See Metcalf (1977) In: Recent Results in Cancer Research 61. Springer-Verlag, Berlin, pp. 1-227.

5VBSTITUTE 5~EE~ (RULE 26 WO 96/33281 PCr/US96/0~432 F,x~mrles The following examples are inrlllded to more fully illustrate the present invention.
Additionally, these examples provide pl er~ d embodiments of the invention and are not 5. meant to limit the scope thereof. Standard methods for many of the procedures described in tlre followrng examples, or suitable alternative procedures, are ~"~,vr~ de~y reo,g~l".ed m~n~ of molecular biology, such as, for Px~mpl~ "Molecular Cloning,"Second Edition (Sambrook, et al., Cold Spring Harbor Laboratory Press, 1987) and"Current Protocols in Molecular Biology" (Ausubel, et al., eds. Greene Associates/Wiley 10 Interscience, NY, 1990).

PREPARATION OF RETROVIRAL VECTOR BAcKsoNEs The following example describes the production of three retroviral vector backbones, decign~ted KT-1, KT-3B, KT-3C. Vector KT-1 differs from KT-3B and KT-3C in that the former lacks a selectable marker which in KT-3B is neomycin resistance, whereas KT-3C
confers phleomycin resi~t~n~e.
The Moloney murine lellk~mi~ virus (MoMLV) S' long terrninal repeat (LTR) EcoR
20 I-EcoR I fr~gm~nt, inrllltiing gag sequences, from the N2 vector (Armentano et al., J. Vir.
61:1647, 1987; Eglitas etal., Science 230:1395, 1985) is ligated into the plasmid SK+
(Stratagene, La Jolla, CA). The resulting construct is deci~n~ted N2R5. The N2R5construct is mllt~ted by site-directed in vitro mutagenesis to change the ATG start codon to ATT preventing gag expression. This mllt~g~ni7e(l fragment is 200 base pairs (bp) in length 25 and flanked by Pst I restriction sites. The Pst I-Pst I mllt~ted fragment is purified from the SK+ plasmid and inserted into the Pst I site of N2 MoMLV 5' LTR in plasmid pUC31 to replace the non-m--t~ted 200 bp fragment The plasmid pUC31 is derived from pUCl9(Stratagene, La Jolla, CA) in which additional restriction sites Xho I, Bgl II, BssH II and Nco I are inserted between the EcoR I and Sac I sites of the polylinker. This construct is 30 de~ign~ted pUC31 /N2R5gM.
A 1.0 kilobase (Kb) MoMLV 3' LTR EcoR I-EcoR I fragment from N2 is cloned into plasmid SK+ resulting in a construct decign~ted N2R3-. A 1.0 Kb Cla I-Hind III
fragment is purified from this construct.

SUBS~mJTE SHEET (RULE 26) The Cla I-Cla I dol.linall~ selectable marker gene fragment from pAFV~I retroviral vector (Kriegler et al., Cell 38:483, 1984; St. Louis et al., PNAS 85:3150, 1988), comprising a SV40 early promoter driving expression of the neomycin (neo) phosphotransferase gene, is cloned into the SK+ pl~emi~l This construct is deeign~ted SK+ SV2-neo A 1.3 Kb Cla I-5 BstB I gene fragment is purified from the SK+ SV2-neo pl~cmitl KT-3B orKT-l vectors are con~,ll u~ d b~ a t~ee part liga~on in wlTich the ~o I-Cla I fragment co~ ;l l;l lg the gene of interest and the 1.0 Kb MoMLV 3' LTR Cla I-Hind m fragment are inserted into the Xho I-Hind m site of pUC31/N2R5gM pl~emi~l This gives a vector ~lecign~te~l as having the KT-1 backbone. The 1.3 Kb Cla I-BstB I neo gene fragment 10 from the pAFVXM retroviral vector is then inserted into the Cla I site of this plasmid in the sense orientation to yield a vector deei n~ted as having the KT-3B backbone.
An alternative selectable marker, phleomycin re~iet~n~-e (Mulsant, et al., Som. Cell andMol. Gen., 14:243, 1988, available from Cayla, Cedex, FR) is used to make theretroviral backbone KT-3C as follows. The plasmid pUT507 (Mulsant, et al., supra) is 15 digested with Nde I and the ends blunted with Klenow polymerase I. The sample is then further digested with Hpa I, Cla I linkers ligated to the mix of fr~ m~nt.c followed by digestion with Cla I to remove excess Cla I linkers. The 1.2 Kb Cla I fragment carrying the RSV LTR and the phleomycin r.oeiet~nce gene is isolated by agarose gel electrophoresis followed by pllrific~tion using Gene Clean (BiolO1, San Diego, CA). This fragment is used 20 in place ofthe 1.3 Kb Cla I-BstB I neomycin r~ciet~n~e fragment to give the backbone KT-3C.

PREPARATION OF RETROVIRAL VECTOR CONSTRUCTS ENCODING PROTEINS

The following example describes the p~ al~LLion of various retroviral vector constructs encoding di~el ~ human genes of interest. More specifically, part (A) describes the production of a vector construct encoding the marker gene ~ galactosidase from E. coli, 30 part (B) human interferon (hIFN), part (C) a retroviral vector construct encoding human interleukin-2 (hIL-2), and part (D) the production of two retroviral vector constructs coding for human factor VIII. The first factor VIII construct, codes for the B domain deleted forrn of the protein while the second construct codes for full length factor VIII

SU~SmUTE SHEE ~ (RULE 26) A. P, t;~,a- ~LIion of CR-~al.

,B-gal is obtained from the plasmid pSP65 as a Hindm-SmaI frR~m~nt s B. Pl ePal ~lion of KT-rhY-IF'N.

To obtain the human ~-IFN gene, the murine homologue is first cloned as follows: A
my-IFl~ cDNA is cloned into the EcoR I site of pUC1813 ~.~s~ntiR11y as set forth below.
Briefly, pUC1813 (co..l~;,.i,~ a sequence encoding ~-~N) is pl~paled as ec.c~ntiRlly described by Kay et al., Nucleic Acids Research 15:2778, 1987; and Gray et al., PNAS
80:5842, 1983) (Figure lA). The m~-IFN cDNA is retrieved by EcoR I digestion of pUC1813, and the isolated fragment is cloned into the EcoR I site of phosphatase-treated pSP73 (Promega; Madison, WI). This construct is de~ignRted SP my-IFN. The orientation ofthe cDNA is verified by a~plo~ liate restriction enzyme digestion and DNA sequ~nr.ing In the sense orientation, the 5' end of the cDNA is R~jac~.nt to the Xho I site of the pSP73 polylinker and the 3' end a~ljac~nt to the Cla I site. The Xho I-Cla I fragment colll~ the my-IFN cDNA in either sense or Rnti~n~e orientation is retrieved from SP my-IFN construct and cloned into the Xho I-Cla I site of the KT-3 retroviral backbone. This construct is de~i~nRterl KT mry-IF'N.
1. Pl ~al ~Lion Of Sequences Encoding h7~-IFN Utilizing PCR

(a) PHA Stimulation Of Jurkat Cells Jurkat cells (ATCC No. CRL 8163) are resuspended at a concentration of 1 x 106 cells/ml in RPMI growth media (Irvine Scientific; Santa Ana, CA) with 5% fetal bovine serum (FBS) to a final volume of 158.0 ml. Phytoh~mo~ggl--tinin ("PHA") (Curtis Mathes Scientific, Houston, TX) is added to the suspension to a final concentration of 1%. The suspension is inc~lb~ted at 37~C in 5% CO2 overnight. The cells are harvested on the following day and aliquoted into three 50.0 ml centrifuge tubes. The three pellets are combined in 50 ml lx phosphate buffered saline (PBS, 145 mM, pH 7.0) and centrifuged at 1000 rpm for 5 minutes The supernatant is decanted and the cells are washed with 50.0 ml PBS. The cells are collected for RNA isolation.
.

SUBSTI~UTE SHEET tRULE 26) WO 96/33281 PCT/US96/0~i432 (~ RNA Isolation The PHA stim~ ted Jurkat cells are resuspended in 22.0 ml ~l~ni~linillm solution (4 M ~l~ni~inillm thiocyanate; 20 rnM sodium acetate, pH 5.2; 0.1 M dithiothreitol, 0.5%
5 sarcosyl). This cell-~ nitlinillm suspension is then passed through a 20 gauge needle six ~mes in order to disrupt cell membranes. A CsCl solution (5.7 M CsCl, O.1 M EDTA) is then overlaid with 11.0 mL ofthe disrupted cell-~l~ni-linil-m solution. The solution is cel~ltrifuged for 24 hours at 28,000 rpm in a SW28.1 rotor (Becl~m~n, Fullerton, CA) at 20~C. After centrifugation the supe~"~ "L is carefully as~ ed and the tubes blotted dry.
~ 10 The pellet is resuspended in a gll~ni~linillm-Hcl solution (7.4 M ~l~ni~linillm-HCl; 25 mM
Tris-HCl, pH 7.5; 5 mM dithiothreitol) to a final volume of 500.0 ~1. This solution is r~" ed to a microcP.ntrifilge tube. Twelve and one-half microliters of concçntrated Glacial acetic acid (HAc) and 250 Ill of 100% EtOH are added to the microfuge tube. The solution is mixed and stored for several days at -20~C to preci~iL~e RNA.
After storage, the solution is centrifuged for 20 minlltes at 14,000 rpm, 4~C. The pellet is then resuspended in 75% EtOH and cçntrifilged for 10 mimltec in a microfuge at 14,000 rpm, 4~C. The pellet is dried by centrifugation under vacuum, and resuspended in 300 L d~ioni~ed (DI) H20. The concentration and purity of the RNA is determined by measuring optical denciti~s at 260 and 280 nm.
(c) Reverse Tr~.3~-,i~lion Reaction Tmme~ t~ly before use, 5.0 l (3.4 mg/mL) of purified Jurkat RNA is heat treated for 5 minutes at 90~C, and then placed on ice. A solution of 10.0 ,ul of lOx PCR buffer (500 25 mM KCI; 200 mM Tris-HCl, pH 8.4; 25 mM MgCl2; 1 mg/ml bovine serum albumin (BSA));
10.0 ~LIoflOmMdATP, 10.0 IlloflOmMdGTP, 10.0 ~loflOmMdCTP, 10.0 ~loflO
mM dTTP, 2.5 ,ul RNasin (40,000 U/ml, Promega; Madison, WIl and 33.0 ~Ll DI H20, is added to the heat treated Jurkat cell RNA. To this solution 5.0 Ill (108 nmol/mL) (Sequence ID No. 1), and 5.0 ,ul (200,000 U/ml) MoMLV reverse transcriptase (Bethesda 30 Research Laboratories, EC 3.1.27.5, MD) is mixed in a microfuge tube and incubated at room temperature for 10 minutes. Follo~,ving the room temperature incubation, the reaction rnixture is incubated for 1 hour at 37~C, and then incubated for 5 rninutes at 95~C. The reverse transcription reaction mixture is then placed on ice in pl~pal~tion for PCR.

SUBSTITUTE SHEET (RULE 26) (d) PCRAmplification The PCR reaction mixture contains 100.0 ,ul reverse transcription reaction; 356.0 l DI
H20; 40.0 ~11 lOx PCR buffer; l.0 ~11(137 nmoVmL) V-OLI #5 (Sequence ID No. 2); 0.5 ~ll (320 nmoVmL) V-OLI #6 (Sequence ID No. 3), and 2.5 ~ll, 5,000 U/ml, Taq polymerase ~EC 2.7.7.7, Perkin-Elmer Cetus, CA). One hundred microlite~; af~s IlllAiUI~ is aliyuut~;d into each of 5 tubes.

(Sequence ID No. l) 5' - 3': TAA TAA ATA GAT TTA GAT TTA
This primer is complel~Glll~ly to a sequence ofthe my-IFN cDNA 30 base pairs do~,vnstream of the stop codon.

V (Sequence ID No. 2) lS 5' - 3': GC CTC GAG ACG ATG AAA TAT ACA AGT TAT ATC TTG
This primer is complementary to the S' coding region ofthe my-IFN gene, beginninP
at the ATG start codon. The S' end of the primer contains a Xho I restriction site.

(Sequence ID No. 3) 5' - 3': GA ATC GAT CCA TTA CTG GGA TGC TCT TCG ACC TGG

This primer is compl~",~ ,., y to the 3' coding region ofthe my-IFN gene, ending at the TAA stop codon. The S' end of the primer contains a Cla I restriction site.
Each tube was overlaid with 100.0 ~11 mineral oil, and placed into a PCR machine25 (Ericomp Twin Block System, Ericomp, CA). The PCR program re~-1~tçc the temperature of the reaction vessel first at 95~C for 1 minute, next at 67~C for 2 minutes and finally at 72~C for 2 minutes. This cycle is repeated 40 times. The last cycle re~l1~tes the temperature of the reaction vessel first at 95~C for l minute, next at 67~C for 2 minutes and finally at 72~C for 7 minntçc The completed PCR amplification reactions are stored at 4~C
30 for l month in preparation for PCR DNA isolation.

(e) Isolation Of PCR DNA

The aqueous phase from the PCR amplification reactions are transferred into a single 3 ~ microfuge tube. Fifty microliters of 3 M sodium acetate and 500.0 ~11 of chloroform:isoamyl SUBST~TUTE SHEET (RULE 26) -WO 96/33281 PCTf~JS96/05432 alcohol (24: 1) is added to the solution. The solution is vortexed and then centrifuged at 14,000 rpm at room temperature for 5 mimltes The upper aqueous phase is transferred to a fresh microfuge tube and 1.0 mL of 100% EtOH is added. This solution is incuh~ted for 4.5 hours at -20~C and then centrifuged at 14,000 rpm for 20 mimltes The supc-~lalall~ is dec~ntetl, and the pellet is rinsed with 500.0 ~LI of 70% EtOH. The pellet is dried by ~ntrifugation ~mder a va~,uulll. The isolated Irf-~FN ~R ~A ~s resu~ell~ed in 10.0 ~I DI
H20.

2. Construction Of h-IFN Retroviral Vectors (a) CreafionAndIsolationOfBlunt-Endedhg-IFNPCRDNAFragments The hr-INF PCR DNA is blunt ended using T4 DNA polymerase. Specifically, 10.0 15 111 of PCR amplified DNA; 2.0 ~11, 10x, T4 DNA polymerase buffer (0.33 M Tris-acetate, pH
7.9, 0.66 M potassium acetate, 0.10 M m~.. e~;.. acetate, 5 mM dithiothreitol, 1 mg/rnL
bovine serum albumin (BSA)); 1.0 ~11, 2.5 mM dNTP (a mixture co.~ g equal molar concentrations of dATP, dGTP, dTTP and dCTP); 7.0 ~I DI H20; 1.0 ~11, 5000 U/mL,Klenow fragment (EC 2.7.7.7, New Fnf~l~n~l Biolabs, MA); and 1.0 ,ul, 3000 U/ml, T4 DNA
polymerase (EC 2.7.7.7, New F,ng]~ntl Biolabs, MA) are mixed together and incubated at 37~C for 15 min--te~, The reaction mixture is then in~ b~ted at room telll~ucl~ re for 40 minutes and followed by an incub~tion at 68~C for 15 min--tes The blunt ended hy-rNF is isolated by agarose gel electrophoresis. Specifically, 2.0 ~11 of loading dye (0.25% bromophenol blue; 0.25% xylene cyanol; and 50% glycerol) is added to reaction mixture and 4.0 ~11 is loaded into each of 5 lanes of a 1 % agarosetTris-borate-EDTA (TBE) gel cont~ining ethidium bromide. Electrophoresis of the gel isperformed for 1 hour at 100 volts. The desired DNA band co.,~ hy-INF, approximately 500 base pairs in length, is vi~ -~li7ed under ultraviolet light.
This band is removed from the gel by electrophoretic transfer onto NA 45 paper (Schleicher and Schuell, Keene, NH. The paper is incubated at 68~C for 40 minutes in 400.0 !11 of high salt NET buffer (1 M NaCI; 0.1 mM EDTA; and 20 mM Tris, pH 8.0) to elute the DNA. The NA 45 paper is removed from solution and 400.0 ~Ll of phenol:chloroform:isoamyl alcohol (25:24:1) is added. The solution is vortexed and centrifuged at 14,000 for 5 minutes. The upper aqueous phase is transferred to a fresh tube and 400 0 ~11 of chloroform:isoamyl alcohol (24:1) is added The mixture is vortexed and SUBS~TUTE SltEEl (RUI E 26) centrifuged for S minutes The upper aqueous phase is ~,an~rel,ed, a second time, to a fresh tube and 700.0 ~11 of 100% EtOH is added. The tube is inc~-b~ted at -20~C for 3 days.
Following incubation, the DNA is pre~ irit~ted from the tube by centrifugation for 20 mimltes at 14,000 rpm. The supe,llaLal~L is ciçç~nted and the pellet is rinsed with 500.0 ~11 of 70%
S EtOH. The pellet, col~ blunt ended hr-IFNDNA, is dried by centrifugation under ~uum and resuspended in 50.0 ~1 of DI H20.
The isolated blunt ended hr-I~;N DNA is phosphorylated using polynucleotide kinase.
Specifically, 25.0 ~11 of blunt-ended h~-IFN DNA, 3.0 1ll of 10x kinase buffer (0.5 M Tris-HCl, pH 7.6; 0.1 M MgC12; 50 rnM dithiothreitol; 1 mM spermidine; 1 mM EDTA), 3.0 ~11 of 10 mM ATP, and 1.0 ~1 of T4 polynucleotide kinase (10,000 U/ml, EC 2.7.1.78, New Fn~l~n~1 Biolabs, MD) is mixed and in~lh~ted at 37~C for 1 hour 45 minutes. The enzyme is then heat inactivated by incub~tin~ at 68~C for 30 min-ltes (~) Ligation Of hr-IFN PCR DNA Into The SK+ Vector An SK+ plasmid is digested with Hinc II restriction endonuclease and purified byagarose gel electrophoresis as described below. Specifically, 5.9 ~1 (1.7 mg/mL).SK+
plasmid DNA (Str~t~ne; San Diego, CA); 4.0 ~11 10x Universal buffer (Stratagene, San Diego, CA); 30.1 111 DI H2O, and 4.0 ~11 Hinc II, 10,000 U/rnL, are mixed in a tube and in~.ubated for 7 hours at 37~C. Following incl~b~tion~ 4.0 ~LI of loading dye is added to the reaction mixture and 4.0 ,ul of this solution is added to each of 5 lanes of a 1% agarose/TBE
gel co~ ethi(1i-~m bromide. Electrophoresis ofthe gel is performed for 2 hours at 105 volts. The Hinc II cut SK+ plasmid, 2958 base pairs in length, is v~ i7~d with ultraviolet light. The digested SK+ plasmid is isolated by gel electrophoresis.
Dephosphorylation of the Hinc II cleavage site of the plasmid is performed using calf intestine alkaline phosph~t~.~e Specifically, 50.0 !ll digested SK+ plasmid; 5.0 ~l 1 M Tris, pH 8.0; 2.0 ~ll 5 mM EDTA, pH 8.0; 43.0 ~l H20 and 2.0 ~l, 1,000 U/mL, calf inte~tin~1 phosphatase ("CIP") (Boehringer ~nnh~im Tndi~n~rolis, IN) are mixed in a tube and incubated at 37~C for 15 min~tes. Following inc~lb~tion, 2.0 Ill CIP is added. and the solution is incubated at 55~C for 90 minutes. Following this incubation, 2.5 Ill 20% sodium dodecyl sulfate ("SDS"), l.0 ~Ll 0.5 M EDTA, pH 8.0, and 0.5 !11, 20 mg;/mL, proteinase K
(EC 3.4.21.14, Boehringer Mannheim, Tn~ n~polis, IN) are added, and the solution is incubated at 55~C for 2 hours. This solution is cooled to room temperature, and 110.0 phenol:chloroform:isoamyl alcohol (25:24:1) is added. The mixture is vortexed and centrifuged at 14,000 rpm for 5 minutes. The upper aqueous phase is transferred to a fresh SUBS~TrUTE S~tEE~ (RULE 26) WO 96/33281 PCI'IUS96/05432 tube and 200.0 ~11 of 100% EtOH is added. This mixture is inr.~lbated at 70~C for 15 mimlt~c. The tube is c~ntrifilged and the pellet is rinsed with 500.0 ~1 of 70% EtOH. The pellet was then dried by centrifugation under a vacuum. The dephosphorylated SK+ plasmid is resuspended in 40 1ll DI H20.
The hr-INF PCR DNA is ligated into the SK+ plasmid using T4 DNA ligase.
Spe~.~fn,ally, 30.0 111 blunt ended, phosphorylated, h~-IFN PCR DNA l~a~ n mixture, 2.0 dephosphorylated SK+ plasmid and 1.0 ,ul T4 DNA ligase are combined in a tube and ins~lb~ted overnight at 16~C. DNA was isolated using a ll.,n~, ep procedure. More specifically, the bacterial strain DH5a (Gibco BRL, Gaithersburg, MD) is transformed with 10 15.0 1ll of ligation reaction mixture, plated on Luria-Bertani agar plates (LB plates) co..l~;..;..g ampicillin and 5-bromo-4-chloro-3-indolyl-13-D-galactoside (X-gal, Gold Biotechnology; St. Louis, MO), and in~1b~tecl overnight at 37~C. DNA is isolated from white bacterial colonies using the procedure described by Sambrook et aL (Mole~ular Cloning, Cold Springs Harbor Press, 1989). The presence of the h y-IFN gene is deLe~ -l,ed 15 by restriction endonuclease cleavage with Xho I, Cla I, Ava II, Dra I, and Ssp I. The expected endonuclease restriction cleavage fragment sizes for pl~erni~le co..l~;..;.~g the hy-IFN gene are presented in Table 2. The isolated DNA plasmid is dçeign~tecl SK h~-IFN and used in constructing the retroviral vectors.

Table 2 Enzyme Fragment Size (bp) Xho I and Cla I 500, 2958 AvaII 222, 1307, 1937 DraI 700, 1149, 1500 Ssp I 750, 1296, 2600 SUBSTITUTE S}~EET (RULE 26) fc) Ligation Of hy-IFN Gene Into Retroviral Vector The interferon gene is removed from SK hy-IFN vector by digestion with Xho I and Cla I restriction endonucleases. The resulting fragment corlt~inin~ the hy-IFN gene is S applox;~ tely 500 bp in length, and is isolated in a 1% agarose/TBE gel electrophoresis.
The Xho I-Cla I hy-~N fragment is then ligated into the KT-3 retroviral backbone. This construct is decign~ted KT hy-IFN. The structure and presence expression of hy-IFN is determined by transforming DH5a bacterial strain ~,vith the KT hy-IFN construct.Specifically, the bacteria is tl~nsrul,l,ed with IS.0 ~1 of ligation reaction mixture. The 10 tl~l~rulllled bacterial cells are plated on LB plates CGl~ ampicillin. The plates are incubated overnight at 37~C and bacterial colonies are selected. The DNA is isolated as described in (b) above, and digested with Xho I, Cla I, Dra I, Nde I, and Ssp I. The expected endonucle~ce restriction cleavage fragment sizes for plasmids containing the hy-IFN gene are presented in Table 3.
Table 3 Enzyme Fragment Size (bp) Xho I and Cla I S00, 6S00 Nde I 1900, S100 Dra I 692, 2700, 3600 Ssp I S41, 1700, 4700 Subsequent sequencing of KT hy-IFN, the retroviral vector, revealed the presence of a one base pair deletion within the hy-IFN gene. This deletion is reversed using multi-step PCR procedure.

i. Sequence Selection Sequences are obtained from IBI Pustell sequence analysis program (Int Biotech, Inc., New Haven, CT).

SUBSTITUTE 5~ T (RULE 26 The following hy-IFN primer sequences are used:

(Sequence ID No. 4) 5'-3': G CCT CGA GCT CGA GCG ATG AAA TAT ACA AGT TAT ATC TTG
5 This primer is the sense sequence col"~ .lPnt~ry to the start codon ATG region P~tPn~ing seven codons upstream of Iry-~N gene, and is ~ecign~ted hr-I~N lb.

(Sequence ID No. 5) 5'-3': GTC ATC TCG TTT CTT TTT GTT GCT ATT
This primer is the anti-sense sequence compl;l 1 l~ Iy to codons 106 to 120 of the hy-IFN
gene, and is dçcign~ted h~-IFN Rep B.

(Sequence ~ No. 6) 51 31: AAT AGC AAC AAA AAG AAA CGA GAT GAC
This primer is the sense sequence compl;ll.. ?.~n.y to codons 106 to 120 ofthe hy-IFN gene, and is decign~ted hy-IFNRep A.

(Sequence ID No. 7) 5'-3': G CAT CGA TAT CGA TCA TTA CTG GGA TGC TCT TCG ACC TCG
This primer is the anti-sense sequence co,llpl;.ll.~ ;..y to the stop codon ATT region and ext~n-ling seven codons U~ lll of the hy-IFN gene, and is design~ted hy-IFN 3b.

ii Initial PCR
A solution of 1 x 106 KT hy-IFN plasmid molecules in 398.0 ,~LI, DI H20; 50 1ll, 10x PCR buffer (500 rnM KCI and 200 mM Tris-HCI, pH 8.4; 25 mM MgC12; 1.0 mg/ml BSA);
5.0 ~LI, 2.5 rnM dATP; 5.0 ~ul, 2.5 rnM dGTP; 5.0 ,ul, 2.5 mM dCTP; 5.0 ~LI, 2.5 mM dTTP;
12.0 ~LI, 18.6 nmol/ml, oligonucleotide hy-IFN lb; 15.0 ~11, 24.6 nmol/ml, oligonucleotide hy-IFN RepB; and 2.5 1ll, Taq polymerase is mixed in a microfuge tube and 50 ,ul is aliquoted into 10 tubes. Similarly, a solution of 1 x 106 KT hy-IFN plasmid molecules in 395.0 !11, DI
H20; 50.0 ~11, lOx PCR buffer (500 mM KCI; 200 rmM Tris-HCI, pH 8.4; 25 mM MgC12; 1 mg/ml BSA);5.0~11, 2.5 mM dATP; 5.0 ~11, 2.5 mM dGTP; 5.0 1ll, ''.5 mM dCTP; 5.0 ~
2.5 mM dTTP; 13 111, 23 4 mnol/ml, oligonucleotide hy-IFN RepA; 17.011 1, 18.0 nmol/ml, oligonucleotide hy-IFN 3b; and 2.5 ~I Taq polymerase is mixed in a microfilge tube and 50 0 SUBS 11 I-lJTE S~EET (RULE 26~

W O 96/33281 PC~rrUS96/05432 ~11 is aliquoted into 10 tubes. The 20 tubes are placed in a PCR m~.-.hine (Model 9600, Perkin Elmer Cetus; Los Angeles, CA). The PCR program re~ tt~c the temperature of the reaction vessel in the first cycle at 94~C for 2 minl-tes The next 35 cycles are re~ ted at 94~C for 0.5 min-ltçc, then at 55~C for 0.5 minlltçe and finally at 72~C for 1 minute. The final cycle is re~ll~ted at 72~C for 10 minl~tes This cycling program is de.cign~ted Program ~0.
Following PCR amplification, 225.0 ~ll of each reaction tube is mixed with 25.0 ~ll loading dye (0.25% bromophenol blue, 0.25% xylene cyanol and 50% glycerol, agarose gel loading dye) and loaded into the wells of a 2% agarose gel co. .~ g ethidil lm bromide.
10 The gel is electrophoresed at appl~-x;---~l~ly 90 volts for 1 hour. Ultraviolet light is used to visualize the DNA band separation. Two bands are isolated, one fragment of 250 bp in size and the other of 150 bp in size by electrophoretic L.al.~rar onto NA 45 paper. Following pl eci~ila~ion, each of the two DNA pellets is resuspended in 20.0 l DI H20 and prepared for further PCR amplification.

iii. Annealing and Second Round PCR

A solution of 20.0 Ill ofthe 150 bp PCRDNA; 20.0 ~11 ofthe 350 bp PCR
20 DNA: 161.5 ~11, DI H20; 25.0 ~LI, lOx PCR buffer (500 mM KCI; 200 mM Tris-HCI, pH 8.4;
25 mM MgCI2; and l mg/ml BSA); 2.5 ~1, 2.5 mM dATP; 2.5 ~1, 2.5 mM dGTP; 2.5 ~11, 2.5 mM dCTP; 2.5 ~ll, 2.5 mM dTTP; and 1.25 ~l Taq polymerase is mixed in a microfuge tube and.47.3 ~1 aliquoted into each of 5 tubes. Each tube is placed in a PCR machine (Model 9600, Perkin-Elmer-Cetus, CA). The PCR program reg-ll~tç~ the temperature ofthe 25 reaction vessel for 5 cycles at 94~C for 0.5 min.ltçs The next cycle is rç~ ted at 55~C for 1 minute. Following this cycle, 1.2 ~ll hy-IFN lb and l.S ~ll hy-IFN 3b are added to the reaction mixture. The tubes are then PCR amplified using program l O. The product is design~fed rhy-IFN.

iv. Creation and Isolation of Blunt-Ended rhg-IFNPCR DNA Fra~ment =~

The PCR amplified hy-IFN DNA is blunt ended using T4 polymerase. Specifically, 120.0 ~ll rh~-IFN PCR solution is mixed with 1.25 lul, 2.5 mM dATP; 1.25 ~ll, 2.5 mM
35 dGTP: 1.25 ~l, 2 5 mM dCTP; 1.25 1, 2.5 mM dTTP; 1 l, T4 DNA polymerase; and I .0 ~l SUBSTITUTE St~EE~ (RULE 2~i3 Klenow fr~mrnt This rnixture is inr.~ ted at room tell,pel~ re for 10 mimltes. Following inr,llh~tion, 13.0 ,ul of agarose gel loading dye is added to the rnixture and this solution is loaded into a 1% agarose gel. The gel is electrophoresed at a~plox;...~lely 90 volts for 1 hour. Ultraviolet light is used to visu~li7e the DNA banding. A 500 bp band is isolated by 5 electrophoretic transfer onto NA 45 paper as described above. Following precipitation, the DNA pellet is resuspended in 12.01 DI ~2O.
The i.eol~ted 500 bp fragment is blunt ended using T4 polynucleotide kinase.
Specifically, 1.0 mg ofthis fragment is mixed with 1.5 ~11 10x kinase buffer (0.5 mM Tris-HCI, pH 7.6; 0.1 mM MgC12; 50 mM dithio~ iLlui; 1 mM sperrnidine; 1 mM EDTA); 1.5 ~1, 10 mM ATP; and 1.0 ~11, T4 polynucleotide kinase, and inr.~b~ted at 37~C for 30 mimltes v. Ligation of rhy -IFNPCR DNA Into the SK+ Vector The rh~-IFN PCR DNA is ligated into the SK+ vector. A solution of 2.0 ~11 h~-IFNPCR DNA-kinase reaction ll~xLule, 2.0 ,ul CIP treated SK+ vector; and 1.0 ~11, T4 DNA
ligase is inr.ub~ted at 16~C for 4 hours. DH5a bacteria is transformed as described above.

vi. Ligation of hy-IFN Gene Into Retroviral Vector Ligation of h~-IFN gene into retroviral vector is performed as described above. The new vector is d~ign~ted KT hy-IFN.

C. Pl epa. ~Lion of KT-hIL-2 .
The method for cloning hIL-2 into KT-3 retroviral vector is essrnti~lly iclentic~l to the procedure for cloning hg-IFN into KT-3, with the exception that di~el~ primers are required for ~mplific~tion of the hIL-2 DNA sequrnre. The following hIL-2 PCR primer sequences are used:
V-OLI #55 (Sequence ID No. 8) 5'-3': ATA AAT AGA AGG CCT GAT ATG
This primer is compliment~ry to a sequence of the hIL-2 cDNA downstream of the stop codon SU8ST~UTE SHEE~ ~RULE Z6) V-OLI #l (Sequence ID No. 9) 5'-3': GC CTC GAG ACA ATG TAC AGG ATG CAA CTC CTG TCT
This primer is the sense sequence ofthe hIL-2 gene comrlim.o.nt~ry to the 5' coding region beginning at the ATG start codon. The 5' end of the primer contains a Xho I
5 restriction site.

V-OLI #2 (Sequence ID No. 10) 5'-3': GA ATC GAT TTA TCA AGT CAG TGT TGA GAT GAT GCT
The primer is the anti-sense seq~l~nce of the hIL-2 gene complimentary to the 3'10 coding region ending at the TAA stop codon. The 5' end ofthe primer cont~inc the Cla I
restriction site.

D. rl ~p~l ~lion of Factor VIII Vectors.
The following is a description of the construction of several retroviral vectorsencoding factor VIII. Due to the size of the full length factor VIII gene (7,056 bp), p~cl~ging ~;onsl~ s of le~lOVil~ll vectors and because selection for tr~n.c~ ced cells is not a requirement for ex vivo hematopoietic stem cell therapy, a retroviral backbone, e.g, KT-l, 20 lacking a selectable marker gene is employed.
A gene encoding full length factor VIII can be obtained from a variety of sources.
One such source is the plasmid pCIS-F8 (EP 0 260 148 A2, published March 3, 1993), which contains a full length factor VIII cDNA whose c,~l es~ion is under the control of a CMV major immerli~te-early (CMV MIE) plomotel and Pnh~n~.~r The factor VIII cDNA2~ contains ~ppl o~ill,ately 80 bp of 5' untr~ncl~ted sequence from the factor VIII gene and a 3 ' untrancl~ted region of about 500 bp. In addition, between the CMV promoter and the factor VIII sequence lies a CMV intron sequence, or "cis" element. The cis element, spanning about 280 bp, comprises a splice donor site from the CMV major immtodi~te-early promoter about 140 bp upstream of a splice acceptor from an immunoglobulin gene, with the30 intervening region being supplied by an Ig variable region intron.

i. Construction of aPlasmid Encodin Retroviral VectorJW-2.

A plasmid, pJW-2, encoding a retroviral vector for expressing full length factor VIIl 3~ is constructed using the KT-l backbone from pKT-I. To facilitate directional cloning ofthe SUBSTIl UTE SHFE~ tRULE 26) factor VIII cDNA insert into pKT- 1, the unique Xho I site is converted to a Not I site by site directed mutagenesis. The resultant plasmid vector is then opened with Not I and Cla I.
pCIS-F8 is digested to completion with Cla I and Eag I, for which there are two sites, to release the fragment encoding full length factor vm. This fragment is then ligated into the 5 Not I/Cla I restricted vector to generate a plasmid desi n~ted pJW-2.

ii. Construction of a Plasmid Encodin~ Retroviral Vector ND-5.

A plasmid vector encoding a truncation of about 80% (appl-~x;, ~IP1Y 370 bp) of the 3' untr~n.Cl~ter~ region ofthe factor vm cDNA, dçcign~ted pND-5, is constructed in a pKT-l vector as follows: As described for pJW-2, the pKT-1 vector employed has its Xho I
restriction site replaced by that for Not I. The factor vm insert is generated by digesting pCIS-F8 with Cla I and Xba I, the latter enzyme cutting 5' ofthe factor VIII stop codon.
15 The appl.,x;,~ ly '7 kb fragment co.,l~ g all but the 3' coding region ofthe factor vm gene is then purified. pCIS-F8 is also ~ estecl with Xba I and Pst I to release a 121 bp fragment co~ g the gene's tf~ ~"h~;on codon. This fragment is also purified and then ligated in a three way ligation with the larger fragment encoding the rest of the factor vm gene and Cla IlPst I restricted BLUESCRIPT~) KS+ plasmid (Strat~ n~ San Diego, CA) to 20 produce a plasmid dçcign~ted pND-2.
The unique Sma I site in pND-2 is then r.h~nged to a Cla I site by ligating Cla I
linkers (New F.ngl~n~l Biolabs, Beverly, MA) under dilute con~itinnc to the blunt ends created by a Sma I digest. After recircularization and lig~tinn, pl~cmi~c co~ two Cla I sites are identified and dçci n~ted pND-3.
The factor VIII sequence in pND-3, bounded by Cla I sites and co"li.;";,~g the full length gene with a truncation of much of the 3' untran.ci~ted region, is cloned as follows into a plasmid backbone derived from a Not I/Cla I digest of pJW-l [a pKT-l derivative by cutting at the Xho I site, blunting with Klenow, and inserting a Not I linker (New Fn~l~n-l Biolabs)], which yields a 5.2 kb Not I/Cla I fr~grnP.nt pCIS-F8 is cleaved with Eag I and Eco RV and the resulting fragment of about 4.2 kb, encoding the 5' portion ofthe full length factor VIII gene, is isolated. pND-3 is digested with Eco RV and Cla I and a 3.1 kb fragment is isolated. The two fr~gTn.ontc cont~ining portions of the factor VIII gene are then ligated into the Not I/Cla I digested vector backbo~e to produce a plasmid d~cign~ted pND-5.

SUBSTJ~UTE S~EET (RUL~ 2~;) iii. Construction of a B Domain-deleted Factor ~III Vector The precursor DNA for the B-deleted FVIII is obtained from Miles Laboratory. This ~A~,ession vector is desi~n~ted p25D and has the exact backbone as pCISF8 above. The S Hpa I site at the 3 ' of the FVIII8 cDNA in p25D is modified to Cla-I by oligolinkers. An Acc 1 to Cla I fragment is clipped out from the modified p25D plasrmd. T~s L~ ,"L spans the B-domain deletion and in~ dçs the entire 3' two-thirds of the cDNA. An Acc I to Cla I
fragment is removed from the pJW-2 above, and replaced with the modified B-domain deleted fragment just described. This construct is desi~n~tell B-del-1.
As those in the art will a,oprecia~e, after construction of ~l~.cmi~ls encoding retroviral vectors such as those described above, such plasmids can then be used in the production of various cell lines from which infectious l~co~ ,illallL lellovil~lses can be produced.

E. Pl ~pa, ~lion of MDR- 1.

Plasrnid clones co..l~ini.~ the the multi-drug rçcict~n~e-l (MDR-l) gene were obtained from ATCC (ATCC No. 61360 and 65704). The gene is isolated and purified using methods provided in Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2d., 1989).
20 Appropriate endonuclease restriction sites, Xho I and Cla I, (Molecular Cloning: ,4 LaboratoryManual, 2d., 1989) are provided for insertion ofthe MDR-l gene into KT-l or KT-3B retroviral vector backbones.

Isolation and Tr~ncrl~lction of Bone Marrow Cells Pluripotent hematopoeitic stem cells, CD34+ are collected from the bone marrow of a patient by a syringe evacuation performed by known techniques. Alternatively, CD34+
30 cells may also be obtained from the cord blood of an infant if the patient is diagnosed before birth. Generally, 20 bone-marrow aspirations are obtained by puncturing femoral shafts or from the posterior iliac crest under local or general an~ctheci~ Bone marrow aspirations are then pooled and suspended in Hepes-buffered Hanks' balanced salt solution con~ining heparin sulfate at 100 Units/ml and deoxyribonuclease I at 100 llg/rnl and then subjected to a 3~ Ficoll gradient separation. The buffy coated marrow cells are then collected and washed SUBSTITUTE SHEET (RULE 26) WO 96/33281 PCI'IUS96/05432 according to CEPRATETM LC (CD34) Separation system (Cellpro, Bothell, WA). The washed buffy coated cells are then stained sequentially with anti-CD34 monoclonal antibody, washed, then stained with biotinylated secondary antibody supplied with the CEPRATE~
system. The cell mixture is then loaded onto the CEPRATE~q avidin column. The biotin-5 labeled cells are adsorbed onto the column while unlabeled cells pass through. The column isthen rinsed according to the CEPRATETM systerrl di~ ,Liolls and C~34+ cells eluted by agitation ofthe column by m~ml~lly squeezing the gel bed. Once the CD34+ cells are purified, the purified stem cells are counted and plated at a concentration of 1 x 105 cells/ml in Iscove's modified Dulbecco's me~ lm, IMDM (Irvine Scientific, Santa Ana~ CA),10 co"~ .g 20% pooled non-heat inactivated human AB serum (hAB serum).
After purification of CD34+ cells, several methods of tr~ned~çin~ purified stem cells may be performed. One approach involves tran.ed~-ctic n of the purified stem cell population with vector co,.l";";l~g supellldl~ll cultures derived from vector producing cells. A second approach involves co-cultivation of an irradiated monolayer of vector producing cells with 15 the purified population of non-adherent CD34+ cells. A third approach involves a similar co-cultivation approach, however the purified CD34+ cells are pre-stim~ ted with various cytokines and cultured 48 hours prior to the co-cultivation with the irradiated vector producing cells. Pre-sfim~ ti-)n prior to tr~neductinn increases t;~;liv~: gene transfer (Nolta et al., Exp. Hematol. 20:1065; 1992). The increased level of tr~ned-lction is attributed to 20 increased proliferation of the stem cells neceee~ry for efficient retroviral traned~lction.
Stimulation of these cultures to proliferate also provides increased cell populations for re-infusion into the patient.

pz~ in~ Cell Production A. MLV structural ~ene expression vectors To decrease the possibility of replication-competent virus being generated by genetic interactions between the MLV proviral vector DNA and the structural genes of the par.k~ging cell line ("PCL"), separate expression vectors, each lacking the viral LTR, were generated to express the gag/pol and env genes independently. To further decrease the possibility of homologous recombination with MLV vectors and the resultant generation of replication-competent virus, minim~l sequences other than the protein coding sequences were used. In SU~3STTTUTE SHE~T (RULE 2~i) order to express high levels of the MLV structural proteins in the host cells, strong transcriptional promoters (CMV e2rly and AdS major late promoters) were utilized. An example of the construction of a MoMLV gag/pol eA~JI es.,ion vector pSCV10 follows:
1. The 0.7 Kb HinCII/XmaIII fragment encomp~ing the human cytomegalovirus (CMV) early transcriptional promoter (Boshart, et al., Cell 41:521, 1985) was isolated.
2. A 5.3 Kb PstI(partial)/ScaI fragment fi~m t~e ~ko~LV ~ ,vi, a~ , MLV-K
(Miller, et aL, Mol. Cell BioL 5:531, 1985) encomp~in~ the entire gag/pol coding region was isolated.
3. A 0.35 Kb DraI fragment from SV40 DNA (residues 2717-2363) encomr~in~
10 the SV40 late transcriptional termination signal was isolated.

4. Using linkers and other standard reco,l,bi l~l,L DNA techniques, the CMV
promoter-MoMLV gag/pol-SV40 termination signal was ligated into the bluescript vector SK+
(Stratagene, San Diego, CA).
An ~ mple of the construction of an MLV xenotropic envelope expression vector 15 follows.
1. A 2.2 Kb NaeI/NheI fragment cr r,~ p the coding region of the xenotropic envelope obtained from clone NZB9-1 (O'Neill, et al., J. Virol. 53: 100, 1985) was isolated.
2. Using linkers and other standard recolllbillal,L DNA teçhni~ s~ the CMV
early promoter and SV40 late tellllillaLion signal described for the gag/pol c~ s~ion above (pSCV10) were ligated in the order CMV promoter-xeno env-terrnination signal (pCMVxeno B. Host Cell Selection Host cell lines were screened for their ability to efficiently (high titer) rescue a drug recict~n~.e retroviral vector A alpha N2 (Arment~n~-, ef al., J. Vir. 61: 1647, 1987; and Eglitas, e~
al., Science 230: 1395, 1985) using replication competent retrovirus to produce the gaglpol and env structural genes ("MA" virus). Titer was measured from confiuent monolayers 16 h after a medium change by adding filtered supernatants (0.45 um filters) to 5x104 NIH 3T3 TK- cells on a 6 cm tissue culture plate in the presence of 4 ug/ml polybrene followed by selection in G418.
Among the non-murine cell lines which demonstrated the ability to package MoMLV-based - vector with high titre were the cell lines CF2 (canine), D17 (canine), 793 (human), and HT1080 (human). These cell lines are pl~felled for production of p~c~ging and producer cell lines, although many other cells may be tested and selected by such means.

SUBSTlTUTE SHEET (RULE 26) CA 022l6868 l997- lO- l7 C. Generation of P~c~ in~ Cell Lines (i) Plc:p~lion of gag/pol int~rmy1i~tçc As examples ofthe generation of gaglpol interm~ tes for PCL production, D17 ~A~C No. CCL-183), 293 (ATCC No. 1573), and ~i11080 (ATCC No. CCL 121) cells were co-tran~fected with 1 ug of the methotrexate r~ei.~t~n~e vector, p~400 (Graham and van der Eb, VirologyS2:456, 1973), and lOugoftheMoMLVgag/poleAl~les:,ionvector, pSCV10 (above) by calcium phosphate co-ple.~ ;on (D17 and HT1080, see Graham and van der Eb, l O supra), or lipofection (293, see Felgner, et al., Proc. Nafl. Acad. Sci., USA 84: 7413, 1987).
After selection for tr~n~fected cells in the presence of the drugs di~ylilllidol and methotrexate, individual drug resistant cell colonies were Pyp~nded and analyzed for MoMLV gaglpol cAI,l ession by extr~c.o.ll~ r reverse transcriptase (RT) activity (modified from Goff, et al., J.
Virol. 38:239, 1981) and intr~c~llnl~r p30gag by Western blot using anti-p30 antibodies (goat antiserum #77S000087 from the National Cancer Tn~titute). This method identified individual cell clones of each cell type which t;A~I essed 10-50x higher levels of both proteins ccJIllp~ ed with that of the p~ ging cell line PA317, as shown in Table 4.

SVBS I lTUTE SHE~ (RULE 26) -- --=
-PROPERTIES OF MoMLV GAG/POL-EXPRESSING CELLS

RT p30gag LARNL
S CELLNAME ACTIVITY (CPM) EXPRESSION TITRE
(CFU/~L) 3T3 800 - N.D.
PA317 1350 +/- 1.2 x 103 D17 800 - N.D.
D17 4-15 5000 ~ 1.2 X 104 D179020 2000 l l l 6.0X 103 D179-9 2200 ~ 1.0X 103 D17 9-16 6100 l l l I l 1.5 X 104 D17 8-7 4000 - N.D.
HT1080 900 - N.D.
HTSCV21 16400 ~ 8.2 X 103 HTSCV25 7900 111 2.8X 103 HTSCV42 11600 ~ 8.0 X 102 HTSCV26 4000 - < 10 293 600 - N.D.
293 2-3 6500 ~ 7 x 104 293 5-2 7600 l l I I l N.D.

The biological activity of these proteins was tested by introducing a retroviral vector, LARNL which expresses both the amphotropic envelope and a Neo+ marker which confers resi.~t~nce to the drug G418. In every case, co-expression of gag/pol in the cell line and env from the vector allowed efficient pac~ging of the vector as determined by cell-free transfer of G418 resistance to 3T3 cells (titer). Titer was measured from confluent monolayers 16 h after a medium change by adding filtered supernatants (0.45 ~lm filters) to 5x104 NIH353 TK+ cells on a 6 cm tissue culture plate in the presence of 4 ug/ml polybrene followed by selection in G418.
- Significantly, the vector titers from the cell lines correlated with the levels of p30gag (Table 4).
Since the level of env should be the same in each clone and is comparable to the level found in PA31 7 (data not shown), this indicates that titre was limited by the lower levels of gag/pol in SUBSTITUTE SHEET ~RUL~ 26) these cells (inrlllrlin~ PA317). The titre correlated more closely with the levels of p30gag than with the levels of RT.

(ii) Conversion of gag/pol lines into xenotropic p~rlr~ging cell lines.
S
As examples of the ~ .aLion of xenotropic PC~s, the gag/pol over-~ OI ~ for D 17(4-15) and HT1080 (SCV21) were co-tr~n~fçcted by the same techniques described above except that 1 ~lg of either the phlec,lllycill re~i~t~nr.e vector, pUT507 (for SCV21), or the hygromycin B le~ ..r.e marker, pY3 (for 4-15, see Blochlinger and DiPP~I...~...~, Mol. Cell Biol. 4:2929, 1984), and 10 ~lg ofthe xenotropic envelope eA~les:,ion vector, pCMVxeno (above) was used. After selection for tr~n~fected cells in the presence of phleomycin or hy~,lulllycin, respectively, individual drug resi~ cell colonies were l~lcp~nllçd and analyzed for intrac~lllll~r eA~Iession of MLV p30gag and gp75e7~v proteins by Western blot using specific antisera. Clones were identified which ~ c;ssed relatively high levels of both gaglpol and xeno 1 5 env.
A number of these xenotropic pacl~ ing cell lines were tested for their capacity to package I ~LI OVil ~I vectors by measuring titre after the introduction of retroviral vectors. The results are plt;sellLed in Table 5, below.

SUBSTITUTE SHEET (RULE 26) VECTOR TITRE ON ~NOTROPIC PCLs KT-1 TITRE (CFIJ/ML) HT1080 SCV21 1.0 x 105 ~1 1.0 x 105 XF7 1.0 x 105 10. ~12 (HX) 4.5 x 105 X6 9.0 X 104 X10 ~DX) 1.3 X 105 X23 8.0 X 104 ~igh~t titers are obtained when retroviral vectors are introduced into pac~ging cell lines by infection, as opposed to transfection (Miller, et al., Somat. Cell Mol. Genel., 12: 175, 1986). However, the xenotropic p~k~ginp~ cell lines described herein are blocked for infection by recolllb;ll~llL xenotropic retroviral particles since the cells express a xenotropic env protein (i.e., "viral interference"). To overcome the problem of "viral interference,"
whereby cell lines expressing a xenotropic envelope protein block later infection by xenotropic MLV vectors able to otherwise infect those cell types, vector particles col~ g other viral envelopes (such as VSV-g protein (Florikiewicz, ef al., J. Cell Bio. 97: 1381, 1983; and Roman, et al., F.xp. Cell ~es 175:376, 1988) which bind to cell receptors other than the xenotropic receptor) may be generated in the following manner. 10 ,ug of the plasmid DNA encoding the retroviral vector construct to be packaged is co-transfected into a cell line which expresses high levels of gag/pol with 10 llg of DNA from which a VSV-g protein is expressed. The resultant vector, cont~ining VSV-g protein, is produced transiently in the co-transfected cells. Two days after transfection, cell free supernatants are added to - prospective xenotropic pack~ing cell lines (which express gag, pol, and em~) Cell free supernatants are then collected from the confluent monolayers and titered by PCR. Cell clones producing the highest titers are selected as pacL-~ging cell lines. This procedure is 3 j sometimes referred to "G-hopping. "

SUB5TlTUTE SHEET (RULE 26) WO 96/33281 PCI~/US96/05432 VII. Alternative Viral Vector p~r.k~in~ Techniques Several additional ~ I;ve systems can be used to produce recombinant retrovirus

5 particles carrying a vector construct according to the invention. Some of these systems take advantage of the fact that the insect virus, baculovirt~s, and the ~ ~ ~ r-- ~ viruses, vaccinia and adenovirus, have been adapted to make large amounts of any given protein for which the corresponding gene has been cloned. For example, see Smith, et al. (Mol. Cell. Biol. 3: 12, 1983); Piccini, et al. (Melh Enymology, 153:545, 1987); and Mansour, et al. (Proc. NatL
10 Acad. Sci. USA 82: 1359, 1985). These and similar viral vectors can be used to produce proteins in tissue culture cells by insertion of appl.~pIiate genes and, hence, could be adapted to make retroviral vector particles.
Adenovirus vectors are derived from nuclear replicating viruses and can be defective.
Genes can be inserted into vectors and used to express proteins in m~mm~ n cells either by in vitro construction (Ballay, et aL, EMBO J. 4:3861, 1985) or by recombination in cells (Th--mm~l, et al., J. Mol. Appl. Geneffcs 1:435, 1982).
One p~ert;Iled method is to construct pl~cm~ using the adenovirus MajorLate Promoter (MLP) driving: (1) gag/pol, (2) env, (3) a modified viral vector construct. A
modified viral vector construct is possible because the U3 region of the 5' LTR, which contains 20 the viral vector promoter, can be replaced by other promoter sequences (~e, for example, Hartman, Nucl. AcidsRes. 16:9345, 1988). This portion will be replaced after one round of reverse transcriptase by the U3 from the 3' LTR.
These pl~cmi~1s can then be used to make adenovirus genomes in vitro (Ballay, et al., supra), which are then tr~n~ft~cted into 293 cells (a human cell line making adenovirus ElA
25 protein), for which the adenoviral vectors are defective, to yield pure stocks of gag/pol, e~.~v and retroviral vector carried separately in defective adenovirus vectors. Since the titers of such vectors are typically 107-10ll/ml, these stocks can be used to infect tissue culture cells ~iml-lt~neously at high multiplicity. The cells will then be programmed to produce retroviral proteins and retroviral vector genomes at high levels. Since the adenovirus vectors are 30 defective, no large amounts of direct cell Iysis will occur and retroviral vectors can be harvested from the cell supernatants.
Other viral vectors such as those derived from unrelated retroviral vectors (e.g, RSV, MMTV or HIV) can be used in the same manner to generate vectors from primary cells In one embodiment, these adenoviral vectors are used in conjunction with primary cells, giving rise to 3~s retroviral vector preparations from primary cells.

SUBSTITUTE SHEE~ (RULE 2~i) Another alternative for making recombinant xenotropic retroviral particles is an in vitro pacL-~ginP; system. For example, such a system can be employ the following components:
1. gag/pol and env proteins made in the baculovirus system in a similar manner as described in Smith, et al., supra, or in other protein production systems, such as yeast 5 or E coli);
2. vector constructs made using T7 or ~P~ L~ ffm systems ~r o~rer suitable in vitro RNA-g~llel~Lillg system (see, for example, Flamant and Sorge, J. ViroL
62:1827, 1988);
3. tRNA made as in (2) or purified from yeast or mAmm~ n cells;
4. liposomes (preferably with embedded env protein); and 5. cell extract or purified components (typically from mouse cells) to provide env processing, and any or other n~c~ssAry cell-derived functions.
Within this procedure, the components of (1), (2), and (3) are mixed. The env protein, cell extract and pre-liposome mix (in a suitable solvent) is then added. In a pl~re,lt:d embodiment, the env protein is embedded in the liposomes prior to adding the resulting liposome-embedded env to the mixture of (1), (2), and (3). The mix is treated (e.g, by sonication, temperature manipulation, or rotary dialysis) to allow encapsidation of the nascent viral particles with lipid plus embedded env protein in a manner similar to that for liposome encapsidation of pharrnAc.euticals, as described in Gould-Fogerite, e~ al., Anal.
Biochem. 148:15, 1985). This procedure allows the production of high titers of replication incompetent recol.,billa"L retroviruses without cc ,-l A"~ A~ ;nn with pathogenic retroviruses or replication-competent retroviruses.

D. Detection of Replication Competent Retroviruses (RCR) The propensity of the Pa~1~Ag;ng cells described above to generate replication competent retrovirus may be stringently tested by a variety of methods, two of which are described below.

i. The Extended S+k- Assay The extended S+L- assay determines whether replication competent, infectious virus is present in the supernatant of the cell line of interest. The assay is based on the empirical observation that infectious retroviruses generate foci on the indicator cell line MiCIl (ATCC
35 No. CCL 64.1). The MiCII cell line is derived from the MvlLu mink cell line (ATCC No.

SUBS 111 LI~E SHEET (RULE 26) CCL 64) by tr~n~d~ction with Murine Sarcoma Virus (MSV). It is a non-producer non-transformed, revertant clone co.,~;,.h~ a replication defective murine sa,-;o.l.a provirus, S+
but not a replication co---~c ,l murine leukPmi~ provirus, L-. Infection of MiCIl cells with replication co"-l,cLc -L retrovirus "activates" the MSV genome to trigger "tran~ru-~aLion"
which results in foci formation.
Su~e~aL~L is lelllu.~ ~nm the cell line ta ~e teste~ lc~ ~ of replicatron competent retrovirus and passed through a 0.45 ~Lm filter to remove any cells. On day 1, Mv1Lu cells are seeded at 1.0 x 1û5 cells per well (one well per sample to be tested) on a 6 well plate in 2 mL Dulbecco's Modified Eagle Medium (DMEM), 10% FBS and 8 ~lg/mL10 polybrene. Mv1Lu cells are plated in the same manner for positive and negative controls on separate6wellplates. Thecellsareinc~ tedovernightat37~C, 10%C02. Onday2, 1.û
mL of test supc --~l~-L is added to the Mv1Lu cells. The negative control plates are incubated with 1.0 mL of media. The positive control consists of three dilutions (200 focus forming units (ffu), 20 ffu and 2 ffu each in 1.0 mL media) of MA virus (Miller, ef al., 15 Molec. and Cell Biol., 5:431, 1985) which is added to the cells in the positive control wells.
The cells are in~lb~ted overnight. On day 3 the media is aspirated and 3 .0 mL of fresh DMEM and 10% FBS is added to the cells. The cells are allowed to grow to confiuency and are split 1:10 on day 6 and day 10, amplifying any replication co.~ L~;-,L retrovirus. On day 13 the media on the Mv1Lu cells is aspirated and 2.0 mL DMEM and 10% FBS is added to 20 the cells. In addition the MiCIl cells are seeded at 1.0 x 105 cells per well in 2.0 mL
DMEM 10% FBS and 8 ~lg/mL polybrene. On day 14 the sup~"~aLallL from the Mv1Lu cells is transferred to the co"e~onding well of the MiCII cells and inc~ ted overnight at 37~C 10% CO2. On day 15, the media is aspirated and 3.0 rnL offresh DMEM and 10%FBS is added to the cells. On day 21, the cells are e~minPd for focus formation (appearing 25 as clustered refractile cells that overgrow the monolayer and remain ~tt~ched) on the monolayer of cells. The test article is d~L~ ed to be co..~ ted with replicationcompetent retrovirus if foci appear on the MiCI l cells.

ii. Cocultivation of Producer Lines and MdH Marker Rescue Assay As an alternate method to test for the presence of RCR in a retroviral particle producing cell line producer cells are cocultivated with an equivalent number of Mus dunni cells (NIH NIAID Bethes~l~ MD). Small scale co-cultivations are performed by rnixing of 5 0 x 105 Mus dunni cells with 5.0 x 105 producer cells and seeding the mixture into 10 cm 3s plates (10 mL standard culture media/plate 4 ~g/mL polybrene) at day 0. Every 3-4 days SUBSmUTE SHEET (RULE 26) W O 96/33281 PC~rrUS96/05432 the cultures are split at a 1 10 ratio and 5 0 x 105 Mus dunni cells are added to each culture plate to effectively dilute out the producer cell line and provide m~imllm amplification of RCR On day 14, culture supt;ll~d~llLs are harvested, passed through a 0 45 ~lm cellulose-acetate filter, and tested in the MdH marker rescue assay Large scale co-cultivations are performed by seeding a mixture of 1 0 x 1 o8 Mus dunni cells and 1 0 x 1 o8 producer cells mto a tatal oftwenty T-150 flasks (30 rnL ~L~ln~l.l culture media/flask 4 ~g;frr~ po~ybrene) Cultures are split at a ratio of 1 10 on days 3, 6, and 13 and at a ratio of 1 20 on day 9 On day 15, the final SupelllaL~llL~i are harvested, filtered and a portion of each is tested in the MdH marker rescue assay The MdH marker rescue cell line is cloned from a pool of Mus dunni cells transduced with LHL, a retroviral vector encoding the l~ygro~ycin B resistance gene (Palmer, et al., Proc. Naf'l. Acad. Sci. USA, 84 1055, 1987) The retroviral vector can be rescued from MdH cells upon infection of the cells with RCR One mL of test sample is added to a well of a 6-well plate co ~ E 1 x 105 MdH cells in 2 mL standard culture merlillm (DMEM with 15 10% FBS, 1% 200 mM L-El~t~min~, 1% non-essenti~l amino acids) co ~ 4 ~g/mL
polybrene Media is replaced after 24 hours with standard culture mo~ m without polybrene Two days later, the entire volume of MdH culture supe --a~ is passed through a 0 45 ~lm cellulose-acetate filter and ~ ~ns~-.ed to a well of a 6-well plate co ~ 5 0 x 104 Mus dunni target cells in 2 rnL standard culture me~ m Col-l ?;~ ~in~ polybrene After 24 20 hours, supe---aLa-.~s are replaced with standard culture media co l~ 250 ,uglmL of hygromycin B and subsequently replaced on days 2 and 5 with media cont~ininp 200 llg/mL
of hygromycin B Colonies resistant to h~g-u~-yci~ B appear and are vic~l~li7ed on day 9 post-selection, by staining with 0 2% Coomassie blue Production of Recombinant Retroviral Particles The production of recombinant xenotropic retroviral particles carrying vector constructs according to the invention, representative examples of which are described above, 30 from the human xenotropic and canine xenotropic p~c~sging cell lines HX and DX, respectively, is described below SUBSTITUTE SHEET (RlJI E 26) A. Transient Plasmid DNA Transfection of p~t~k~in~ Cell Lines HX and DX

The p?~r~ ing cell line HX or DX is seeded at 5 0 x 105 cells on a 10 cm tissue culture dish on day 1 with DMEM and 10% fetal bovine serum (FBS). On day 2, the media is replaced with 5.0 mL fresh media 4 hours prior to ~,~nsre.,Lion. Standard calcium p~p~ate-DNA CO-pleCi~ila~ions are pelrullllC~ by mixing 40.0 1112.5 M Cack, 10 ~g of the plasmid encoding the vector to be packaged, and deionized H2O to a total volume of 400 ~1. The DNA-CaC12 solutions are then added dropwise with constant agitation to 400 1ll of p~eci~iL~ion buffer (50 mM HEPES-NaOH, pH 7.1; 0.25 M NaCI and 1.5 mM Na2HPO4-10 NaH2P04). These lllixLulèS are in~ b~ted at room temperature for 10 minlltes The resultant fine precipitates are added to di~elellL culture dishes of cells. The cells are incubated with the DNA precipitate overnight at 37~C. On day 3, the media is aspirated and fresh media is added. Sup~ x are removed on day 4, passed through 0.45 ~lm filters, and stored at -80~C.
B. Pa~ in~ Cell Line Tr~n~ ctinn DX or HX p~ ging cells are seeded at 1.0 x 105 cells/3 cm tissue culture dish in 2 ml DMEM and 10% F~3S, 4 ~g/mL polybrene (Sigma, St. Louis, MO) on day 1. On day 2, 20 3.0mL, 1.0mLand0.2mLofeachofafreshlycollectedsu~ ",.~ Collli1;ll;l~g VSV-g pseudotyped retroviral particles car~ying the desired vector are added to the HX cells. The cells are inc~b~ted overnight at 37~C. On day 3, the pools of cells are cloned by limiting dilution by removing the cells from the plate and counting the cell suspension, diluting the cells suspension down to 10 cells/mL and adding 0.1 rnL to each well (1 cell/well) of a 96 25 well plate (Corning, Corning, NY). Cells are incubated for 14 days at 37~C, 10% CO2.
Several clones producing the desired lecollll~h~ xenotropic retrovirus are s~lected and exp~n~ed up to 24 well plates, 6 well plates, and finally to 10 cm plates, at which time the clones are assayed for eAIJleS~iOn ofthe ap~.up,iate retroviral vector and the supernatants are collected and assayed for retroviral titer.
Using the procedures above, DX and HX cell lines may be derived that produce recombinant xenotropic retroviral vectors with titers greater than or equal to 1 x 1 o6 cfil/mL
in culture.

SUBS~ITUTE 5HEEl' tRULE 2~i) C. Titer Assays Normally vector titers are determined by tr~n.cd~lctiQn oftarget cells such as HT1080, with applupliate dilutions of a vector plcpar~lion, followed by antibiotic selection and counting of surviving colonies (WO 91/02805). However, recombinant retroviral vectors carrying a desired vector construct may not include a ger~e c~c~fora s~ , 5~, asmay be the case when the vector construct encodes a large gene of interest, for instance, full length factor VIII, titering assays other than those based on selection of drug resistant colonies are required. To this end, antibody and PCR assays, the latter of which is described below, may be employed to determine retroviral vector titer, i.e., the number of infectious particles comprising the retroviral vectors of the invention. While such a PCR assay may be required in the context of a vector lacking a selectable marker, it is understood that such an assay can be employed for any given vector.
To use PCR to amplify sequences unique to the retroviral vectors of the invention, various primers are required. Such primers can readily be ~leci n.od by those skilled in the art and will depend on the retroviral vector backbone employed and the components thereof, the particular region(s) desired to be amplified, etc. Representative examples of particular primer pairs include those specific for LTR sequences, pa~ ing signal sequences or other regions of the retroviral backbone, and also include primers specific for the gene of interest in the vector. Additional advantages in using such a PCR titering assay include the ability to assay for genome rearr~ng~m~nt, etc.
In the practice of the present invention, the PCR titering assay is performed bygrowing a known number of HT1080 cells, typically 1 x 10~ cells, tr~nc~llced with a retroviral vector capable of dil e~;~i.,g CA~ ssion of the gene of interest on 6-well plates for at least 16 hr. before harvest. The retroviral vectors used for these trancd~ctions are preferably obtained from cell culture supe"-aLal-l~. One well per plate is reserved for cell counting.
Cells from the other wells are Iysed and their contents isolated. DNA is prepared using a QIAmp Blood Kit for blood and cell culture PCR (QIAGEN, Inc., Chatsworth, CA). DNAs are resuspended at 5 x 1 o6 cell equivalents/mL, where one cell equivalent is equal to the DNA content of one cell.
To calculate titer, a standard curve is generated using DNA isolated from untransduced HT1080 cells (negative control) and IIT1080 cells tran.cduced with a known vector and having one copy of that vector per cell genome (positive control), such as may be prepared from packaging cell lines tr~n.c~uced with a retroviral vector encoding a selectable 3 5 marker. e.g., neomycin resistance. For both the positive and negative controls, DNA is SUBSTITUTE SHEET (RULE 26) W O 96/33281 PC~rrUS96/05432 resuspended at 5 x 1 o6 cell equivalents/mL. The standard curve is generated by cOlllbil~illg di~re~ l amounts of the positive and negative control DNA, while keeping the total amount of DNA constant, and amplifying specific sequences th~.~fio", by PCR using primers specific to a particular region of the retroviral vector. A representative group of rnixtures for S generating a standard curve is:

Tube 100% 75% 50% 25% 10% 5% 0% Blank Positive Control (~lL) 50 37.5 25 12.5 5 2.5 0 0 Negative Control (,uL) 0 12.5 25 37.5 45 47.5 50 0 10 Distilled water (~lL) 0 0 0 0 0 0 0 50 5.0 ,uL from each tube is placed into one of eight reaction tubes (duplicates are also prepared), with the r~n~inder being stored at -20~C. 5.0 IlL from each sample DNA
~l~alaLion are placed into their own reaction tubes in ~ plie~te. PCR reactions (50 ~L total 15 volume) are then initi~ted by adding 45.0 ,uL of a reaction mix co,.~ . the following components per tube to be tested: 24.5 ~lL water, S IlL 10X reaction PCR buffer, 4 ,uL of 25 mM MgCI2, 4 ~lL dNTPs (co~ i..g 2.5 mM of each of dATP, dGTP, dCTP, and dTTP), S ~L of primer rnix (100 ng or each primer), 0.25 IlL TaqStart monoclonal antibody (Clontech Laboratories, Inc., Palo Alto, CA), 1.00 ~lL TaqStart buffer (Clontech Labs, Inc.), 20 and 0.25 ~LL AmpliTaq DNA polymerase (Perkin-Elmer, Inc., NorwaLlc, CN). Just prior to aliquoting the reaction mix to the reaction tubes, 1 ~lL of a-32P dCTP (250 ,uCi; 3000 C/mmol, 10 mCi/mL, Amersham Corp., Arlington Heights, IL) is added into the reaction mix. After aliquoting 45 .0 ~lL the reaction rnix into each of the reaction tubes, the tubes are capped and placed into a thermocycler. The particular den~lul~ion, ~nn~ling, elongation 25 times and temperatures, and number of thermocycles will vary depending on size and nucleotide composition of the primer pair used. 20 to 25 ~mplific~tion therrnocycles are then pel Çol l"ed. 5 ~L of each reaction is then spotted on DE8 1 ion ~ch~n~e chromatography paper (Wh~trn"n Maidstone, F.n~l~nd) and air dried for 10 min. The filter is then washed five times, 100 mL per wash, in 50 rnM Na2PO4, pH 7, 200 mM NaCl, after which it is air 30 dried and then sandwiched in Saran Wrap. Qll~ntit~tion is performed on a PhosphoImager SI (Molecular Dynamics, Sunnyvale, CA). Filters are typically exposed to a phosphor screen, which stores energy from ionizing radiation, for a suitable period, typically about 120 min. After exposure, the phosphor screen is sc~nnP-1 whereby light is emitted in proportion to the radioactivity on the original filter. The sc~nnin~ results are then downloaded and 3~ plotted on a log scale as cpm (ordinate) versus percent positive control DNA (abscissa).

SUE~STITUTE SHEE~ (RULE 26) , Titers (infectious units/mL) for each sample are calculated by multiplying the number of cells from which DNA was isolated by the percentage (converted to decimal form) determined from the standard curve based on the detected radioactivity, divided by the volume of retroviral vector used to tr~n.cduce the cells. As will be appreciated by those in the art, other S methods of detection, such as colorimetric methods, may be employed to label the amplified pFoducts.

Lar~e Scale Production of R.eco"~bil,allt Xenotropic Retroviruses The recombinant retroviruses of the invention can be cultivated in a variety of modes, such as in a batch or continuous mode. In addition, various cell culture technologies can be employed to produce commercial scale qu~ntiti~ ofthe l~col"bin~,~ retroviruses according 15 to the invention. Several such techniques are described below, although others known to those in the art may likewise be employed.

A. Reco,l,billa"l Retrovirus Production From Hollow Fiber Cultures i. Cul~ure Initiation To initiate a hollow fiber culture, the hollow fiber bioreactor (e.g, H~B; Cellco, Inc., Germantown, ~) is first conditioned for 48 hours prior to seeding by ~im~ tinp a run condition with 100-200 mL of complete growth media at 37~C. The growth media 25 preferably is that to which the cell line has been adapted. All liquids in the HFB when originally shipped should be aspirated and replaced with the complete growth media. When seeding the bioreactor, the cells should not have been split more than 48 hours earlier and should be in log growth phase at the time of harvest for the seeding of the ~B. The cells typically are harvested by trypsinazation and pelleted by centrifugation. The cell pellet is 3 0 then resuspended in 4 mL of 25% pre-conditioned media and delivered to the extra-capillary space by syringe using the side syringe ports found on the H~B. After seeding the E~B, the cells are allowed to adhere for 20 to 30 minutes before starting the circulation pump. During this time, the media used to condition the ~B is replaced with 100-200 mL of 25% pre-conditioned media. The circulation feed pump is initiated with the starting flow rate set at 25 35 mL/min. (setting 5 with 2 long pump pins). After 1 hour from the time of switching the SVBSTlTUTE SHEET (RULE 26) pump on, a one mL sample of media is collected in order to record the initial levels of lactate and ammonia. On a daily schedule, 1 ml samples are collected every 24 hours to assay for the daily production of lactate and ammonia. The initial 100-200 mL of media is .oY~.h~nged with fresh media when lactate levels begin to reach 2.0 g/L (or the equivalent to 22 mM/L).
5 The same volume of media is replaced until the culture approaches daily levels of 20 mmol/L.
~}ren daily levels of lactate reach 20 mmol/L, the size of the reservoir bottle is ~ ~ to a 500 rnL bottle c~,..l;.;..;.~g 500 r~L offresh media. The fiow feed rate is then increased to 50 mL/min. when the culture begins to produce 2.2 mmol/day of lactate. When daily 500 mL
volumes reach 20 mmol/L of lactate, the original Cellco supplied reservoir feeding cap is ~ cl~ ed for a larger reservoir cap (Unisyn-vender part #240820) adapted for the Cellco system with the addition of tubing and male luer lock fittings. This reservoir cap will accommodate 2 liter Corning bottles. (To avoid the exchange of reservoir caps during a culture run, initiate the run with a large reservoir cap which can also support smaller bottle sizes.) When daily lactate readings are assayed and recorded, the daily levels of lactate production ofthe culture can be used to determine when the culture reaches m~ximllm cell density, i.e., when the rate of lactate decreases and levels o~

ii. Seeding Densitv for 7~he 2X-,~-gal To establish specific seeding requi~ ~,lL:i, two hollow fiber runs are performed, one run seeded with a low number of cells, the other seeded with a high number of cells.
Progress of each culture is tracked by analyzing the daily glucose consumption and lactate production levels.
In this experiment, one HFB was seeded with 1.3 x 107 cells (-~p~ese~.l;..g the low seed culture), the other with 1.6 x 108 cells. Here, the cell line 2x-13-GALl7 l4 was able to initiate a good hollow fiber run under both seeding conditions. Tniti~ting a run with fewer cells is primarily convenient for red~lr.ing the effort required for generating the number of cells required to start a culture, although fewer cells initially extends the time it takes to reach optimal cell densities, which usually yield the highest titers. 2x-J3-GAL17 l4 adapted well to hollow fiber culture, eventually requiring daily media changes of 500 mL in order to avoid P~cc~lm~ tion of toxic levels of lactate. Pl~te~llinp; of daily lactate production and drops in peak titer production correlated with maximum cell densities and the relative health of the culture.

iii. Optimal TiterConcen7rations, FrequencvofHarvestsand To~al SUBSTITUTE SHEET (RULE 26) WO 96/33281 PCI'IUS96/05432 Harvesf Amounfs 13-gal titers for the above experiment were determined from frozen samples on 293 cells assayed 48 or 72 hours after tr~n~duction. The tr~n~ ced cells were stained for ~3-gal 5 activity and counted on a hemocytometer to yield a titer based on the number of blue cells /mL (BCT/mL). Optimum titers were generally obtained on day 7 af ~ ~r see~ cllrlture at 1.8 X108 BCT/mL from a 72 hour blue cell titer on 293 cells. A duplicate culture initially seeded at a 10 fold lower seeding density peaked at 5.2 x107 BCT/mL from a 48 hour blue cell titer. Cc~l"pa~ ~d to flat stock cultures (from tissue culture dishes or flasks) titered using 48 hour blue cell titers on HT1080 cells (c~lc~ ted to be about 5 X106 BCT/mL), the increase in titer by using hollow fiber systems is applu,~;,..~t.oly ten fold higher. These m~imllm titers observed were reached prior to hitting 20 mmoVL lactate levels, which appeared to reduce titers produced the following week.
Crude suptorn~t~ntc can be harvested every 9 hours with out any loss of titer and 15 three harvests per day should be possible with minimllm titre loss. In addition, continuous hollow fiber cultures can be ..l~ d for several weeks. When titers were co."pa~t:d between the low and the high seed culture, there was little differences by day 11 between the two seed cultures, both of which averaged 4 x 107 BCT/mL.

Two-Phase Purification of Recol"bil~a"L Retroviruses A Concentration of DA/ND-7 recombinant particles 1400 ml of media (DMEM co~ g 5% Fetal Bovine Serum) CO"I;.;";"g DX/ND-7 vector at a titer of 1.25 x 106 cfu/ml is used as starting material. Three hundred millilit-ors of two-phase partitioning components (PEG-8000 (autoclaved), dextran-sulfate, and NaCI) are added to a final concentration of 6.5% PEG, 0.4% dextran-sulphate, and 0.3 M NaCl. The 30 resultant solution is placed into a two-liter separatory funnel, and le~ in a cold room for 24 hours (including two mixing steps approximately 6 to 16 hours apart).
- Following the 24 hour period, the bottom layer (applo~l.laLely 20 mL) is carefully eluted, and the interphase (approximately 1 mL) is collected in a 15 mL conical FALCON
tube. The interphase cont~ining vector is diluted to 10 mL by addition of PBS, and SUBSTITUTE SHEET (RULE 26) in~ b~ted at 37~C in order to bring the solution to room temperature and destabilize the mic~llec To one-half of the diluted interphase, KCI is added to a final concentration 0.4 M, and mixed well. The tube is then placed on ice for ten mimltes, and spun for 2 minutes at 2,000 rpm in a bench-top c~ntrifilge. The sup~ is removed and filtered through a 0.45 ,um syringe filter. The other ~}a~of the interphase col l~ il-g vector is se~a~ ed by S-500 Seph~ Y cl~ llla~ography in lX PBS. The results of these concentration processes, as determined in a BCFU assay, are shown below in Table 6:

PHASE QUANTITY OF VECTOR
Crude 1.75 x 109 bcfu Separation: Top phase 1.4 x 108 bcfu Separation: Interphase 7(+/-3) x 108 bcfu Separation: Bottom phase 2 x lo6 bcfu Final step: KCI separation *6(+/-3) x lo8 bcfu Final step: S-500 separation *1.8(+/-0.3) x 108 bcfu * Note that since the sample was split into two halves, that these numbers were doubled in order to represent the level of purification that would be expected if the entire 1 mL
interphase was separated as indicated.
ln summary, 1.4 liters of crude research grade supelll~L~nl co.ll~;llil-g recolllbhlallL
retroviral particles may be reduced to a 10 mL volume, with applu~illl~Lely 50% (+/-20%) being recovered when KCI separation is utilized as the final step. When S-500 chlulll~Lography is utilized as the final step, only about 10% ofthe initial recombinant retroviral particles are recovered in a 14 mL.
In order to complete concentration of the retroviral vector particles, the vector-cont~ining solution may be further subjected to concentration utili7ing an MY-membrane Amicon filter, thereby reducing the volume from 10 to 14 mL, down to less than 1 mL.

SUBST~TUTE St~EET (RULE 26) Production of Vector from DX/ND7 ,~-gal Clone 87 Utilizing a Cell Factory DX/ND7 bgal clone 87, an ~A~ ssion vector, was grown in cell factories. Cells were ~rown in DMEM supplemented with Fetal Bo vi..~; ~er~rrr irr rol~erbat~es u~t~ err~g~r ~Is to seed 20 10-layer cell factories (NUNC) at a 1:3 dilution were obtained. Each 10-layer cell factory is seeded with apploX;~ y 0.8 liters of cell medium.
Cells were seeded into the cell factory by pouring media co..l~ g cells into the10 factory so that the suspensions evenly fill the 10 layers. The factory is then carefully tilted away from the port side to prevent the suspension from redistribution in the common tube.
Finally, the cell factory is rotated into its final upright position. A hepavent filter is ~tt~r.h~d to each port. The factory was then placed in a CO2 incubator.
In three days, and for each ofthe next three days, supt;lllatal,L co~l;.;..;..g vector was 15 harvested. The cell factory is placed in a tissue culture hood. One filter is removed and sterile Ll~ rel tubing is conntocted to the open port. The factory is lifted so that supelllaL~I-L
drains into the tubing. Applox;...~l~ly 2 liters of sup~ ..l is harvested from each factory.
Fresh DMEM~FBS is used to replenish the lost me~ m The transfer tubing is removed and the factory replaced in the inr.~lb~tor. From 20 cell factories, ~l c Ainlately 90 liters of crude 20 vector cn--l;~ g supelllaL~IL were obtained.
Verification of the vector was performed by tr~n~duction of HT 1080 cells. These cells were harvested 2 days later and stained for b-gal protein. The titer of the supernatant was determined to be 2 x l 07/ml.

Concentration of Recolllbilla.lL Retrovirus by Low-Speed Centrifugation A. Retrovector Supernatant Pl t;pa~ ~Lion Producer cell lines DA/13gal and HX/DN-7 were cultured in a culture flask and a roller bottle, respectively, cont~inin~ Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum plus lmM L-Glutamine, Sodium pyruvate, non-ec.c~nti~l amino acids and antibiotics. Viral supe--laLal.L was harvested from the flask and SUBST~UTE SHEET ~RULE 26) , WO 96/33281 PCT/US96/0~432 roller bottle, and were filtered through a 0.45 um syringe filter. The filtered supeln~La were stored either at 4~C (~/ND7), or frozen at -70~C (DA13-gal).

B. Virus Concentration Viral ~up~lllaL~lL was aliquoted into 50 ml ster~e OAKR~D~E screw cap tubes, placed into an SS34 rotor for use in a Sorvall cPntrifil~e The tubes were spun for 1 hour at 16,000 rpm (25,000g-force) at 4~C. Upon completion ofthe spin, the tubes were removed, the sup~ L~IL clec~nte(l and a small opaque pellet resuspended in the DMEM media10 described above.

C. Virus Titration ConcellLl~L~d virus was titered on HT1080 cells plated 24 hours earlier at a cell 15 density of 2xl 05 cells per well in a six well plate + 4 ,ug/ml polybrene. Briefly, virus preps were diluted from 1/10 to 1/10,000 and 50 111 of each dilution was used to infect one well from the six well plate. Plates were incllb~ted overnight at 37~C. Forty-eight hours later, cells were fixed and stained with X-gal. The results are set forth below in Table 7.

Table 7. Virus Concentration through Low Speed Centrifugation ~ , number Parameter descrip~ion 1 2 3 Virus source DAB-gal DA~-gal ~tND7 DAB-gal HX/ND7 Titer of normal harvest 4.4 x 10 6 Z.l x 10 6 3.2 x 10 5 5x106 5x105 Titer of virus con. ~ 6X108 7.4x107 3.2x107 2.9x108 3.9x107 Starting volume 80 ml .39 rr~ 39 ml 118ml 40ml FiIIal collce~ Le volu~ne .5 ml .36 rnl .36 rnl 78ml .28rnl Fold virus concentration 136X 34X lOOX 58X 78X
Virus recover~ 87% 30% 91% 50% 99%

SUBSTITUTE SH EET (RULE 26) As is evident from Table 7, virus recovery ranged from 30% to 99%, with the bestrecovery being obtained from human producer cells (HX/ND7; recovery ranged from 91% to 99%).

Concentration of Recombinant Retroviruses By Ultrafiltration S-SOO purified supelllal~llL col-lh;..i..~ the ¦3-gal ~;A~lt;ssing recombinant retrovirus 10 DX/CB-bgal and partially col-cellLlal~:d ,u~ellla~ co~ the same virus were each filtered through a 0.45 um filter, and loaded into a CENTR~PREP-100 filter (product #4308, Amicon, MA). The supelllaL~l~, were kept at a temperature of 4~C throughout thisprocedure, in~ lrlin~ during c~ntrifi~g~tion. The CENTRIPREP filters were spun three times each for 45 to 60 minutes at 500 x G. Between each spin the filtrate was dec~nted The 15 retent~te was thus sequentially reduced, such that the initial 15 mL (or 10 mL) volume was reduced to apprc~x;.~ (ely 0.6 rnL per unit.
The resultant titer was determined by assaying HT1080 target cells set up at a concentration of 1 x 105 cells per well 24 hours prior to tr~n.~dllctinn of the viral sample.
Cells were tr~n.cduced in the presence of 8 ,ug/ml polybrene and 2 rnL growth media (DMEM
20 plus 10% FBS) per well. As shown in Table 8 below, appro,~ ely one hundred percent of the virus was recovered lltili7in~ this procedure (note that titers are in BCFU/ml).

Table 8 Pre-cG,.I"r" G~J Final titer/volume. titer/volume S-500 4 x 107/15 ml 1.3 x 109/0.6 ml parL conc. 3 x 108/10 ml 1 x 101~/0.6 ml Preparation of Recombinant Retrovirus in a Bioreactor - 30 A. Freezin~; protocol SUBSTITUTE 5~tEE~ tRULE 26) Producer cells are frozen in DMEM media co~ i";.~,o 10% to 20% FBS, and S to 15% DMSO, at a concentration of 1 x 107 cells/ml/vial. Cells are frozen in a controlled rate freezer (Series PC, Controlled Rate Freezing System, Custom Biogenic Systems, Warren Ml) at a rate offrom 1 to 10~C per minute. Frozen cells are stored in liquid nitrogen.
B . Bioreactor pn~tocol Cells are thawed from frozen vials at 37~C, washed once with media to remove DMSO, and ~Yp~n~ed into 850 cm2 "FALCON" roller bottles (Corning, Corning, NY) Fxp~n~ed cell culture is used to inoculate a "CELLIGEN PLUS" bioreactor (5 liter working 10 volume; New Brunswick, Edison, NJ). The cells are grown on microcarriers (i.e., Cytodex 1 or Cytodex 2; Pharmacia, Pisca~w~y, N.J.) at a concentration of 3 to 15 g/L microcarrier.
Initial inoculation d~n.eiti~s are from 4 to 9 cells/bead at halfto full volume for 2 to 24 hours.
The media cnnetit~nte for virus production are DMEM-high glucose (Irvine Scientific, Santa Ana, CA.) basal media supplem~nterl -with FBS (10 to 20%), cTlllt~min~ (8 to 15mM), glucose (4.5 to 6.5 g/L), Non~oss~nti~l amino acids (1O, RPMI 1640 amino acids ( 0.2 to 9.6X), 10 mM HEPES, RPMI 1640 Vitamins (0.2 to 5X).
During culture, pH (6.9 to 7.6) and dissolved oxygen ("DO" 5 to 90%) are controlled by the use of a four gas system which inrllldes air, oxygen, nitrogen, and carbon dioxide. After several days of batch growth the culture is then continuously perfused ~,vith 20 fresh media with concurrent continuous harvesting in an esc~l~tin~ perfusion rate of 0.5 to 2.5 volumes/day. Cell retention is the result of difrt~ Lial se~iimPnt~tion of cell covered beads in a dec~nting column.
During operation the bioreactor is monitored for viable cells, titer, glucose, lactate, ammonia levels, and lack of co..~ ..;. .Al ;on. Viable cells and titer range from 1 x 105 cells/ml to 1 x 107 cells/ml. Glucose ranges from 6 to 0.25 g/L, Lactate from 1 to 25 mM, and Ammonia ranges from 0.5 to 30 mM. Cells are in~lb~ted in the bioreactor for 5 to 25 days.

Cell sortin~ and analysis Apheresed samples were obtained with informed consent from multiple myeloma patients treated at the University of Arkansas Medical Center. The patients were treated on day l with cyclophosphamide at 6 g/m2 (1.5 g/m2 every 3 hours x 4 doses). From day l until 3 5 the start of leukopheresis (usually 10-28 days), granulocyte macrophage colony Stimnl~ting SUB5TITUTE SHEET (RULE 26) factor (GM-CSF) was given at 0.25 mg/m2/day. Apheresis for total white cells was started when the peripheral blood white cell count was greater than 500 cells/ml and the platelet count was greater than 50,000 cells/ml. Patients were apheresed daily until from 6 x 108 mon- mlçle~r cells (MNC) were collected.
S Antibodies to CD14 and CD15 were obtained as FITC conjugates from Becton-~nson. Antibody to Thy-1 (GM201) was o~ ned f~om E~r. Woffl~Tg ~ettrg ~dwrg Tn.etitutç, New York), and was ~etecte(l with anti-yl-PE conjugate from Caltag. Antibody to CD34 (Tuk 3) was obtained from Dr. Andreas Ziegler (University of Berlin), and detected with an anti-y3-Texas Red conjugate (Southern Bioterhnologies).
For cell sorting, fresh MPB samples were elutriated with a JE5.0 Beckman counterflow .?ll-tri~tor equipped with a Sanderson chamber (Bec~m~n, Palo Alto, CA). Cells were resuspended in elutriation m~ m (Biowhittaker, Walkersville, MD) at pH 7.2,supplem.~nted with 0.5% human serum albumin (HSA). The rotor speed was set at 2000 RPM, the cells were introduced, and the first fraction collected at a flow rate of 9.6 ml/min.
15 Fractions 2 and 3 were collected at the respective fiow rates of 14 and 16 mVmin. The larger cells le~ ;np~ in the chamber were collected after stopping the rotor. Cells were resuspended in RPMI suppl~m~nted with 5% HSA, 10 ,ug/ml DNAse I and penicillin/streptomycin at 50 U/ml and 50 llg/ml, respectively. Fractions 2 and 3 were pooled and inr.~ ted with 1 mg/ml heat-inactivated human gamma-globulin to block non-20 specific Fc binding. Granulocytes were further depleted by inc~b~tinn with CD15 conjugated to magnetic beads (Dynal M450, Oslo, Norway) followed by m~gnt-tic selection.
Anti-CD34 antibody or an IgG3 isotype m~t~.hed control were added to cells in staining buffer (HBSS, 2% FCS, 10 mM HEPES) for 20 minutes on ice, together with anti-Thy- l antibody at 5 mg/ml. Cells were washed with a FCS underlay, and then incnb~ted 25 with Texas Red conjugated goat anti-mouse IgG3 antibody and phycoerythrin-conjugated goat anti-mouse IgGl antibody for 20 minutes on ice. Blocking IgGl was then added for 10 minutes. After blocking, the FITC-conjugated lineage antibody panel (CD14 and CD15) was added, and incubated for another 20 minutes on ice. A~er a final washing, cells were resuspended in staining buffer cont~ining propidium iodide (PI).
Cells were sorted either on the FACSTAR Plus cell sorter equipped with dual argon ion lasers, the primary laser emittinf~ at 488 nm and a dye laser (Rhodamine 6G) emitting at 600 nm (Coherent Innova 90, Santa Cruz, CA) or on a high speed cell sorter as described in PCT patent application number PCT/US93/08205.. Residual erythrocytes. debris and dead cells were excluded by light scatter gating plus an FL3 (PI) low gate. Following isolation of SUBSmUTE ~;~EET (RULE 26) WO 96/33281 PCr/US96/OS432 a cell population by fiow cytometry, the sample was diluted 1: 1 in HBSS, pelleted, and resuspended in HBSS for hemocytometer counting.

Tr~n~-luction 1 x 105 CD34+Thy+Lin~ (MPB) viable cells obtained as described above, were suspended in 1 ml of ~eshly thawed retroviral sup~llla~lL with cytokines at the following concentrations: c-kit ligand (Amgen) 100 nglml; IL-3 (Sandoz) 25 ng/ml; IL-6 (Sandoz) 50 10 ng/ml. PloL~I~ e sulfate was added at a final concentration of 4 ug/ml. At 24 and 48 hours, supernatant was replaced with freshly thawed retroviral supernatant. Cytokines and pl~Lalllille sulfate were added at the concentrations listed above. After 72 hours, cells were harvested and placed in assays to determine tran.cductit n frequency. As a control, cells were cultured DMEM with cytokines and plo~ e sulfate as described above, but without 15 retroviral supell.al~lL.

Methylcellulose assay In order to determine tr~nc~ ction frequency of the stem cells, the following experiment was performed. 5 x 103 or 2.5 x 103 cells from each tr~ncd~lction were added to 5 ml of methylcellulose (Stem Cell Technologies) co. .l ~;..;. .~ the following cytokines: c-kit ligand 10 ng/ml; GM-CSF 25 ng/ml; G-CSF 25 nglml; IL-3 10 ng/ml; rhEPO 2 units/ml. 1.1 ml of the cell/cytokine methylcellulose mixture was plated onto four 3 cm gridded plates using a 5 ml syringe and 16.5 gauge needle, and the plates were placed in a 37~C incubator for 2 weeks.
After 14 days, single methylcellulose colonies were picked and suspended in 50 ~11 Lysing Buffer (75 mM KCI, 10 mM Tris-HCI pH 9.25, 1.5 mM MgC12, 0.5% Tween 20, 0.5% NP40, 1 mg/ml proteinase K) for PCR analysis.
The Iysates were amplified by PCR to determine the presence of the vector in thetransduced cells. The PCR assay amplified a 134 bp fragment of the vector psi p?(ck~ging sequence A 40 cycle amplification (25 ul total volume) in Perkin-Elmer 9600 Cycler using 10 ul lysate and 0.20 uM each F1 and B5 primers (Genset) was performed as follows: one cycle 95~C 30 sec.; 40 cycles 95~C 10 sec, 64~C 15 sec., 72~C 15 sec.; final extension 72~C

SU8S I I I UTE SHEET (RULE 26) CA 022l6868 l997- lO- l7 - 81 ~

5 min. PCR products were vi~ li7ecl on ethidium bromide agarose gels. The results appear in Table 9.

F. ~ ~ Tissue(Batch) Titer Colonies Tr~

1 M F-7Xeno(8/18194)1.0 x 107 90 69 77%
1 MF-7D M E M Mock 20 1 5%
2 M F-13Xeno (8118194) 1.8 x 106 80 S 6%
2 M F-13Xeno(8118194)1.0 x 107 86 41 48%
3 #9748Xeno (8118194) 1.0 x 107 86 48 56%
4 M F-20Xeno(8118194)1.8 x 106 81 25 31%
4 M F-20Xeno(8118/94)1.0 x 107 81 39 48%
M F-27Xeno (8118194) 1.8 x 106 81 5 6%
M F-27Xeno (8118194) 1.0 x 107 81 25 31%

6 M F-30Xeno (8118194) 1.8 x 106 81 3 4%
6 M F-30Xeno (8118194) 1.0 x 107 81 35 43%

7 ~DF-13Xeno(8/18/94)1.0 x 107 70 18 26%

8 ~DF-10Xeno(8/18/94)1.0 x 107 70 19 27%

Example lS
LTCIC Transduction Assay CD34+Thy+Lin- (MPB) viable cells were counted and tran~rltlced for 72 hours as lS described in Example 13 herein. Viable cells seeded in 2-fold serial dilutions in a 96- well plate on pre-formed AC6.21 monolayers in Whitlock-Witte media (50/50 RPMI/IMDM, SU8S TUTE ~i~EET ~RULE 2~i) 10% Fetal Calf Serum, Pen-Strep. L-Gl~lt~minç, Sodium Pyruvate, and 2-~) supplem.onted with LIF at 50 ng/ml and IL-6 at 10 ng/ml. Final cell concentrations, set up in duplicate, were 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 cells/well respectively. The plates were cultured at 37~C, 5% CO2 for 5 weeks, with the cells fed weekly by repl7~c~m~-nt of one-half 5 the spent media with fresh media and cytokines.

Bulk LTCIC Assay:

After 5 weeks, plates were scored for cobblestone area forming cells (CAFC).
10 LTCIC plates were prepared by harvesting wells co~ g multiple cobblestones and filtering the cultures through Nitex filters into 24-well plates. Cutlures were overlaid with a~ ox;lll~ly 0.6 mls methylcellulose col-l~;..il-g 10 ng/ml c-kit ligand, 25 ng/ml GM-CSF, 25 ng/ml G-CSF, 10 ng/ml IL-3, and 1.2 U/ml rhEPO, and cultured for 10-14 days at 37~C, 5% CO2 (i.e., a~>plvxul~alely 7 weeks culture from the time oftr~n~ ction). Individual 15 methylcellulose colonies were picked and analyzed by PCR for the presence of the vector psi pacl~gin~ sequence as described in F.Y~mple 14 herein. The results are shown in Table 10 below. The numbers ~e~,~sen~ the number of bulk LTCIC wells with at least one PCR
positive colony/total number of bulk wells analyzed by PCR. The results show that the oLlvpic vector did tr~n~ -ce stem cells as measured by LTCIC. The numbers do not20 represent a tr~n~ -ction frequency of LTCIC since the wells that were harvested contained multiple cobblestone areas, and the resulting methylcellulose colonies could have been derived from di~e,~ cobblestone area forrning cells and hence LTCIC.

Clonal LTCIC Assay:
The clonal LTCIC assay was perforrned as described above for the bulk LTCIC assay with the exception that individual wells were harvested where, based on lirniting dilution, the wells harvested were most likely colonal (>95% probability based on Poisson distribution.
i.e, <37% of the wells at a given dilution contained a CAFC) SUBSTITUTE St~EET (RULE 26) The results are shown in Table 10 below. The numbers represent the number of clonogenic LTCIC wells with at least one positive colony by PCR/total number of clonal wells analyzed by PCR. The results from one t~ lent show that the xenotropic vector transduced 2 of 9 S LFCIC. Since the cells from each well harvested were denved ~o~n 2 single cell, the results of the clonal LTCIC assay ~ l c;se~s a true LTCIC tr~n.cd~lction frequency.

E~periment R~ Titer Bulk LTC-IC Clonal LTC-IC
1. Xeno 1.8e6 0/4 ND
2. Xeno 1. 8e6 0/3 ND
3. Xeno 1.8e6 2/12 2/9 4. Xeno le7 1/7 ND
10' Numbers intlic~te no. bulk LTCIC wells with at least one psi(+) positive colony/total number of bulk wells anaylzed by PCR.

** Numbers indicate no. clonogenic LTCIC wells with at least one psi(+) positive colony/total number clonal wells analyzed by PCR.
While the present invention has been described above both generally and in terms of ~-erelled embo~lim.o.nt~ it is understood that variations and modifications will occur to those skilled in the art in light of the description, supra. Therefore, it is intended that the appended claims cover all such variations coming within the scope of the invention as cl~imed Additionally, the publications and other materials cited to illllmin~te the background of the invention, and in particular, to provide additional details concerning its practice as described in the detailed description and examples, are hereby incorporated by reference in their entirety.

SUBSTITUTE SHEE~ (RULE 26) CA 022l6868 l997-l0-l7 WO96/33281 PCTrUS96/05432 ~h~U~N~ LISTING
(1) GENERAL lN~ TION:
(i) APPLICANTS: CHIRON VIAGENE, INC.
SYSTENIX
(ii) TITLE OF lNv~NllON: High Ef~iciency Ex Vivo Transduction o~
~ ~opoietic Stem Cells by Recn-~nAnt Xenotropic Retro~iral Prepara~ons (iii) N~ RR~ OF .';~':yUl.:N('~ 0 (iv) CORRE~uN~N~ PnD~R-cS:
'A ADDRESSEE: Chiron Viagene, Inc.
,B STREET: 4560 Horton Street ~C CITY: Emeryville ~D STATE: Cali~ornia ,E~ COUN-1'KY: U,S.A.
~,F, ZIP: 94608 (v) C~_~ul~ READABLE FORN:
'A) NEDIUN TYPE: Floppy disk 'B) CO_~U-1~: IBN PC compatible ,C) OPERATING SYSTEN: PC-DOS/NS-DOS
~,D) SOFTWARE: PatentIn Release X1.0, Version ~1,25 (vi) ~u~R~N-l APPLICATION DATA:
(A) APPLICATION NnMRR~: Unassigned (B) FILING DATE: 19 April 1996 (C) CLASSIFICATION:
(~riii) A'1-1U~CNl:Y/AGENT lN~O.~-L TION:
(A) NANE: Kruse, Norman J.
(B) REGISTRATION NnMRR~: 35,235 (C) R~N~/DOCKET NU~BER: 1156,100 (ix) TEL~C~-~ IN I CATION lN~O.~L TION:
(A) TELEPHONE: (510) 601-3520 (B) TELEFAX: (510) 655-3542 (2) lN~-O~LATION FOR SEQ ID NO:1:
(i) ~yu~N~ CHAR~rTR~TSTICS:
'A) LENGTH: 21 base pairs ~B) TYPE: nucleic acid ~C) STRANnRnPR-~S: single ,D) TOPOLOGY: linear (ii) NOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TAATA~ATAG ATTTAGATTT A 21 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single SUBSTITUTE SH EET (RULE 26) W 096/33281 PCTrUS96/05432 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
r (xi ) ~U~N~ DESCRIPTION: SEQ ID NO:2:
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(Xi ) ~QU~'N~ DESCRIPTION: SEQ ID NO:3:
GAATCGATCC ATTACTGGGA TG~l~l-l~A CCTGG 35 (2) INFORNATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
~'A) LENGTH: 40 base pairs ~B) TYPE: nucleic acid ~C) STRANn~nNFCS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) ~U~N~h DESCRIPTION: SEQ ID NO:4:

(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GTCATCTCGT 'll~l-l-l-l-l~l TGCTATT 27 r (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid SUBSTITUTE StlEET tRuLE 26) W O96/33281 PCTrUS96/05432 (C) STRPNnRnNRqS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(Xi ) ~yU~N~ DESCRIPTION: SEQ ID NO:6:

(2) lN~O~L~TION FOR SEQ ID NO:7:
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(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE cHARprTR~TqTIcs ~A) LENGTH: 21 base pairs B~ TYPE: nucleic acid C STRPN~RnNRSS: single ,D TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
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(i) ~yu~N~ CHARACTERISTICS:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

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GaATCG~ITT AT~AAGTCAG TGTTGAGATG ATGCT ~5 SUBSTmJTE SHEET ~RULE 26)

Claims (45)

WE CLAIM:

1. A method of producing transduced hematopoietic stem cells, the method comprising:
(a) obtaining a population of hematopoietic stem cells from a patient; and (b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest.

2. The method of Claim 1 wherein the gene of interest encodes a protein or an active portion of a protein selected from the group consisting of a cytokine, a colony stimulating factor, a clotting factor, and a hormone.

3. The method of claim 1 wherein said recombinant retroviral particle is a xenotropic recombinant retroviral particle.

4. The method of claim 1 wherein said hematopoietic stem cell is a CD34+Thy-1+Lin-hematopoietic stem cell.

5. The method of claim 1 wherein said recombinant retroviral vector particles are a high titer preparation of recombinant retroviral particles.

6. A composition comprising a population of hematopoietic stem cells transduced with recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest.

7. The composition of Claim 6 wherein the gene of interest encodes a protein or active portion of a protein selected from the group consisting of a cytokine, a colony stimulating factor, a clotting factor, and a hormone.

8. The composition of Claim 7 wherein said cytokine is selected from the group consisting of IL-2 and gamma-interferon.

9. The composition of Claim 6 wherein said recombinant retroviral particle is a xenotropic recombinant retroviral vector particle.

10. The composition of Claim 6 wherein said hematopoietic stem cell is a CD34+Thy-1+Lin- hematopoietic stem cell.

11. The composition of claim 6 wherein said hematopoietic stem cells are transduced with a high titer preparation of hematopoietic stem cells.

12. A hematopoietic stem cell transduced with a recombinant retroviral particle substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particle carries a vector construct encoding a gene of interest.

13. The hematopoietic stem cell of Claim 12 wherein the gene of interest construct encodes a protein or active portion of a protein selected from the group consisting of a cytokine, a colony stimulating factor, a clotting factor, and a hormone.

14. The hematopoietic stem cell of claim 12 wherein said hematopoietic stem cell is a CD34+Thy-1+Lin- hematopoietic stem cell.

15. The hematopoietic stem cell of claim 12 wherein said recombinant retroviral vector particle is a xenotropic hematopoietic stem cell.

16. The hematopoietic stem cell of claim 15 wherein said hematopoietic stem cell is transduced by a high titer preparation of recombinant retroviral particles.

17. A method of treating a patient having a genetic disease, the method comprising:
(a) obtaining a population of hematopoietic stem cells from the patient;
(b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating the genetic disease; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced hematopoietic stem cells.

18. The method of claim 17 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles.

19. The method of claim 17 wherein said hematopoietic stem cells are CD34+Thy-1+Lin-hematopoietic stem cells.

20. The method of claim 17 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles.

21. A method of treating a patient having cancer, the method comprising:
(a) obtaining a population of hematopoietic stem cells from the patient;
(b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating cancer; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced hematopoietic stem cells.

27. The method of claim 21 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles.

23. The method of claim 21 wherein said hematopoietic stem cells are CD34+Thy-1+Lin-hematopoietic stem cells.

24. The method of claim 21 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles.

25. A method of treating a patient having an infectious disease, the method comprising:
(a) obtaining a population of hematopoietic stem cells from the patient;
(b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating the infectious disease; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced hematopoietic stem cells.

26. The method of claim 25 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles.

27. The method of claim 25 wherein said hematopoietic stem cells are CD34+Thy-1+Lin-hematopoietic stem cells.

28. The method of claim 25 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles.

29. A method of treating a patient having a degenerative disease, the method comprising:
(a) obtaining a population of hematopoietic stem cells from the patient;
(b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating the degenerative disease; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced hematopoietic stem cells.

30. The method of claim 29 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles.

31. The method of claim 29 wherein said hematopoietic stem cells are CD34+Thy-1+Lin-hematopoietic stem cells.

32. The method of claim 29 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles.

33. A method of treating a patient having an inflammatory disease, the method comprising:
(a) obtaining a population of hematopoietic stem cells from the patient;
(b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating the inflammatory disease; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced hematopoietic stem cells.

34. The method of claim 33 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles.

35. The method of claim 33 wherein said hematopoietic stem cells are CD34+Thy-1+Lin-hematopoietic stem cells.

36. The method of claim 33 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles.

37. A method of treating a patient having a cardiovascular disease, the method comprising:
(a) obtaining a population of hematopoietic stem cells from the patient;
(b) transducing the population of hematopoietic stem cells with recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating the cardiovascular disease; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced hematopoietic stem cells.

38. The method of claim 37 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles.

39. The method of claim 37 wherein said hematopoietic stem cells are CD34+Thy-1+Lin-hematopoietic stem cells.

40. The method of claim 37 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles.

41. A method of treating a patient having an autoimmune disease, the method comprising:
(a) obtaining a population of hematopoietic stem cells from the patient;
(b) transducing the population of hematopoietic stem cells with recombinant xenotropic retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant xenotropic retroviral particles carry a vector construct encoding a gene of interest useful in treating the autoimmune disease; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced hematopoietic stem cells.

42. The method of claim 41 wherein said population of hematopoietic stem cells is transduced by a high titer preparation of recombinant retroviral particles.

43. The method of claim 41 wherein said hematopoietic stem cells are CD34+Thy-1+Lin-hematopoietic stem cells.

44. The method of claim 41 wherein said recombinant retoviral vector particles are xenotropic recombinant retroviral vector particles.

45. The method according to any one of claims 17, 21, 25, 29, 33, 37 or 44 further comprising expanding the transduced population of hematopoietic stem cells in vitro prior to re-introduction of the cells into the patient.